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El debate ético y de regulación sobre eluso de CRISPR-Cas9 en la línea germinal humana

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http://www.scielo.org.co/pdf/unmed/v62n4/2011-0839-unmed-62-04-206.pdf

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Científicos chinos aseguran haber creado los primeros bebés “genéticamente editados”

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Los debates se han centrado en general en las consecuencias sociales, bioéticas y legales del uso de la tecnología de edición del genoma en las células germinales humanas. Los científicos en general están de acuerdo en que CRISPR-Cas9 debe ser permitido para su uso en la creación de modelos de enfermedades humanas, y en la comprensión del desarrollo y los mecanismos moleculares de las enfermedades; sin embargo, debe ser prohibido con fines de eugenesia o mejora. Cuando las cuestiones éticas, las preocupaciones de seguridad, y las dificultades de la aplicación se consideran juntas, se predice que la edición terapéutica del genoma en embriones humanos no será posible en el futuro próximo.

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Abstract

Genome editing technologies have led to fundamental changes in genetic science. Among them, CRISPR-Cas9 technology particularly stands out due to its advantages such as easy handling, high accuracy, and low cost. It has made a quick introduction in fields related to humans, animals, and the environment, while raising difficult questions, applications, concerns, and bioethical issues to be discussed. Most concerns stem from the use of CRISPR-Cas9 to genetically alter human germline cells and embryos (called germline genome editing). Germline genome editing leads to serial bioethical issues, such as the occurrence of undesirable changes in the genome, from whom and how informed consent is obtained, and the breeding of the human species (eugenics). However, the bioethical issues that CRISPR-Cas9 technology could cause in the environment, agriculture and livestock should also not be forgotten. In order for CRISPR-Cas9 to be used safely in all areas and to solve potential issues, worldwide legislation should be prepared, taking into account the opinions of both life and social scientists, policy makers, and all other stakeholders of the sectors, and CRISPR-Cas9 applications should be implemented according to such legislations. However, these controls should not restrict scientific freedom. Here, various applications of CRISPR-Cas9 technology, especially in medicine and agriculture, are described and ethical issues related to genome editing using CRISPR-Cas9 technology are discussed. The social and bioethical concerns in relation to human beings, other organisms, and the environment are addressed.

Keywords: Genome editing, CRISPR-Cas9 technology, bioethical issues, bioethics

1. Introduction

For many years, molecular biologists have sought ways to use cellular repair mechanisms to manipulate DNA through genome editing. In this way, they would have the power to change the genome by correcting a mutation or introducing a new function (Rodriguez, 2016). For this purpose, genome editing technologies were developed (Memi et al., 2018). In recent years, clustered regularly interspaced short palindromic repeats technology (CRISPR-Cas9) has become the most preferred method of gene editing. This technology has advantages such as high accuracy, easy handling, and relatively low cost compared to previous technologies, such as zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN). Thanks to these benefits, CRISPR-Cas9 technology can be easily applied in any molecular biology laboratory.

Genome editing technologies are used in the formation of human disease models in experimental animals and for the understanding of basic gene functions. They also have great therapeutic potential for future treatment of untreated diseases such as certain cancers, genetic disorders, and HIV/AIDS. Today, genome editing in somatic cells is one of the promising areas of therapeutic development (Otieno, 2015). However, various bioethical issues have arisen due to the potential impact of these technologies on the safety of food stocks and clinical applications (Hundleby and Harwood, 2018; Hirch et al., 2019). This review discusses the challenges, possible consequences, and bioethical issues of CRISPR-Cas9 in detail.

2. Biology and function of CRISPR-Cas9 technology

Genome editing technologies often work by creating fractures in chromosomal DNA. ZFN, TALEN, and CRISPR-Cas9 are all based solely on nucleases (Kim and Kim, 2014; Roh et al., 2018). The strength of these technologies stems from the ability to create fractures in the desired region of a specific target sequence as determined by the researcher. This allows researchers to modify the genome in practice in any region (Memi et al., 2018).

The creation of changes in the genome depends mainly on the DNA repair capacity of the cells (Lau et al., 2018). All cells have two basic mechanisms for the repair of double chain breaks on DNA. One of them is nonhomologous end joining (NHEJ) and the other is the homologous dependent repair (HDR) mechanism. In NHEJ, the ends of the fractures are quickly connected directly to each other, regardless of the sequence homology, while HDR requires homology to repair the damaged DNA site. In order to achieve homology, the undamaged sister chromatid is used as a template and DNA damage is repaired (Urnov, 2018).

CRISPR-Cas9 is a naturally occurring defense system in prokaryotic organisms that provides resistance to foreign genetic elements such as plasmids and bacteriophages (Barrangau et al., 2007; Marraffini and Sontheimer, 2008). When the virus or plasmid enters a bacterial cell, CRISPR-Cas9 allows the addition of short viral DNA molecules to the CRISPR site. CRISPR sequences (CRISPRs) are short DNA repeats of viral or plasmid origin found in the genomes of bacteria and are defined as clustered regularly interspaced short palindromic repeats. Cas genes (CRISPR-related) are genes that encode nuclease or helicase proteins associated with CRISPR repeat sequences that have the function of cutting or dissolving DNA (Jansen et al., 2002). Cas9, a member of the Cas gene family, was isolated from Streptococcus pyogenes and is an endonuclease capable of cutting DNA from two active cut regions at both ends of the DNA double helix (Doudna and Charpentier, 2014; Rodriguez, 2016). The CRISPR-Cas system recognizes the DNA of the invading virus or bacterium and directs the Cas protein to destroy foreign DNA (Otieno, 2015).

In the following years, it was discovered that the CRISPR-Cas system can be programmed to find and cut specific target DNA regions, thereby providing genome editing (Jinek et al., 2012; Hsu et al., 2013). As a result of understanding that the human genome can be edited by CRISPR-Cas9, it became clear that genome editing could also be used for therapeutic purposes, and a new era in genetic engineering began (Lau et al., 2018; Roh et al., 2018).

3. Application areas

3.1. Animal models

CRISPR-Cas9 can be used to create animal models to mimic human diseases and to understand disease development by mutating or silencing genes. A mouse model has been developed to determine the harmful effects of mutations in cancer by making mutations that cause the loss of function in tumor suppressor genes or give functions to protooncogenes (Chin, 2015).

Conventional genetically modified (GM) mouse models are produced by gene targeting in embryonic stem cells or transgenesis, which are time-consuming and highly expensive. With CRISPR-Cas9, GM mice can be efficiently produced in a much shorter time (Mei et al., 2016). It can be applied to nonhuman primates such as monkeys. Nonhuman primates are more similar to humans in anatomical, physiological, and genetic terms than rodents (Zhang et al., 2014). Therefore, they are more suitable models than rodents in understanding human biology and disease development (Xin et al., 2016). The first successful application of CRISPR-Cas9 in nonhuman primates, from which a knockout monkey was produced, was realized in 2014 (Niu et al., 2014). However, genome sequences of many nonhuman primates are not yet fully identified. This makes it difficult to design selected single-guide RNAs (sgRNAs) (Gou and Li, 2015; Lou et al., 2016). Therefore, the application of CRISPR-Cas9 in nonhuman primates is still at an early stage.

3.2. Genome editing in specific tissues

Researchers have been able to modify the genomes of specific tissues such as liver and brain tissues using hydrodynamic injection and adeno-associated virus (AAV) (Rodriguez et al., 2014; Senis et al., 2014). In a study, CRISPR-Cas9 has been successfully and effectively applied to the mammalian nervous system. A mixture of green fluorescent protein (GFP)-labeled AAV-spCas9 and AAV-spGuide plasmids was transferred in vivo to the hippocampal toothed brain folds of adult male mice (Swiech et al., 2015). It is thought that the number of such applications will grow in the fields of cancer and neuroscience in the following years (Mei et al., 2016).

3.3. Multiple gene mutations

CRISPR-Cas9 can be used to generate mutants for target genes. In the first such study by Li et al., six sgRNAs targeting Cas9 mRNA and six different genomic regions encoding the Tet1, Tet2, and Tet3 genes were transferred to the cytoplasm of rat embryos (Li et al., 2013). Findings showed that all three Tet genes carried the desired mutations in 59% of newborn rat pups. Successful results were also reported in studies with zebrafish embryos and Arabidopsis as well (Ota et al., 2014; Wang et al., 2015).

3.4. Epigenome studies

Epigenome studies can be performed in two different ways: genome or epigenome editing (Chen et al., 2014; Huisman et al., 2015). In genome editing, nuclease is used to modify the DNA sequence, whereas in epigenome editing, an effector domain is used and the DNA sequence is not changed. This function is achieved by catalytic inactivation of the Cas9-associated effector domain by replacing the Cas9 protein. Altered effector proteins are used to activate or suppress transcription (Lau and Davie, 2016). In epigenome editing, the epigenome can be modified by changing the proteins that maintain and protect the epigenome. Suppression of DNA methylation as a result of degradation of catalytic domains that accelerate the loss of spherical DNA methylation in human cells and lead to cell death is a good example of epigenome studies using CRISPR-Cas9 (Liao et al., 2015).

Another application is the editing of long nonencoded RNAs (lncRNAs) and enhancer RNAs (eRNAs) that can control gene expression and epigenome processes. In a study, eRNA-expression factors and IncRNA-expression enhancers were suppressed by stimulating deletion mutations in a lymphoma cell line using CRISPR-Cas9 (Pefanis et al., 2015). It is predicted that CRISPR-Cas9 may allow different levels of epigenome modification and facilitate further changes to humans (Liao et al., 2015; Mei et al., 2016).

3.5. Treatment of diseases

CRISPR-Cas9 can be applied to cells in vivo or ex vivo. In the in vivo approach, CRISPR-Cas9 is directly transferred to cells in the body using either viral or nonviral methods. In the ex vivo approach, first the cells are removed from the body; then CRISPR is applied to the cells and they are transferred back to the body (Roh et al., 2018). This approach has great potential to develop tissue-based therapies (Rath et al., 2015). Using CRISPR-Cas9, the mutation in the dystrophin protein responsible for the most common form of Duchenne muscular dystrophy was successfully removed (Amoasii et al., 2018; Duchêne et al., 2018; Koo et al., 2018; Long et al., 2018). There are studies to prevent and treat AIDS by inhibiting the entry of HIV into the cell or by removing the HIV genome integrated into the host genome using CRISPR-Cas9 (Saayman et al., 2015). Induced pluripotent stem cells (iPSCs) were successfully produced from cystic fibrosis patients with confirmed F508 deletion in the cystic fibrosis transmembrane regulator ( CFTR) gene by CRISPR-Cas9 (Firth et al., 2015). There are also studies for cataracts (Wu et al., 2015; Yang et al., 2016) and Parkinson's disease (Yang et al., 2016). However, recent studies have shown that CRISPR-Cas9 activates the type 1 interferon (INF) pathway, causing a type 1 INF-mediated immune response (Kim et al., 2018; Charleswort et al., 2019). These findings currently limit the use of CRISPR-Cas9 in treatment.

3.6. Industrial uses

CRISPR was first used for commercial purposes to make bacterial cultures used in cheese and yogurt production resistant to viral infections (van Erp et al., 2015). One of the applications in agriculture is to produce GM crops (Hundleby and Harwood, 2019). There are attempts to increase the yield in the livestock industry (van Erp et al., 2015). It can be used to control invasive pest species to reverse pesticide and herbicide resistance in insects and weeds or to prevent disease spread (Esvelt et al., 2014). Researchers have succeeded in preventing the spread of genes protecting mosquitoes from harmful malaria parasites (Gantz et al., 2015) and making female mosquitoes infertile in the laboratory (Hammond et al., 2016). Vaccine development is another significant area of interest. The smallpox virus vector (VACV) is used in the eradication and vaccination of smallpox. Using CRISPR-Cas9, the efficiency of marker-free VACV vectors has been increased (Yuan et al., 2015). Another example is the hepatitis B vaccine. In order to prevent viral gene expression and replication, specific regions of the hepatitis B genome were targeted and cut by CRISPR-Cas9 (Ramanan et al., 2015).

3.7. RNA editing

Single-stranded RNA (ssRNA) sequences can also be edited by CRISPR-Cas9. In RNA editing, CRISPR-Cas9 consists of a DNA oligonucleotide presenting the PAM (protospacer adjacent motif) region (PAMmer), ssRNA, guide RNA (gRNA), and Cas9 protein. PAMmer acts as a PAM region specifically recognized by Cas9 and directs Cas9 to bind and cut the target ssRNA. 5'-Elongated PAMmers containing bases paired with different ssRNAs and immediately in front of PAM are required for specific binding of target ssRNAs. Since RNA molecules have different functions than DNA, CRISPR-Cas9 can offer a much more flexible application than other genome editing methods (Mei et al., 2016).

3.8. Military applications

One of the lesser-discussed application areas of CRISPR-Cas9 technology is its use for military purposes. As is known, a substantial portion of genome editing studies are supported by the defense ministries of the countries. These studies are commonly focused on increasing the tolerance of soldiers against biological or chemical warfare. This technology has the potential to influence human performance optimization (Greene and Master, 2018). Studies are usually concentrated on discovering different genes that can be harnessed from other species (Gracheva et al., 2010) and identifying new genes that can be associated with posttraumatic stress disorder, which is frequently experienced by soldiers (Cornelis et al., 2010). In a study by Zou et al. (2015), researchers developed dog embryos with higher muscle mass using CRISPR-Cas9. Another interesting study showed that the CMG2 gene, known to cause low sensitivity to anthrax toxin when expressed in small amounts, could be silenced by this technology (Arévalo et al., 2014). However, it should be noted that far more research needs to be conducted for using CRISPR technology in humans as a defense tool against biological and chemical weapons (Greene and Master, 2018).

3.9. DNA replacement in human embryos (germline genome therapy)

The most controversial usage of CRISPR-Cas9 is the modification of human embryo DNA, or, in other words, its use for germline genome therapy. In 2015, a group of Chinese researchers led by Junjiu Huang applied CRISPR-Cas9 to remove a mutation that causes β-thalassemia, which is a fatal blood disease, from the human β-globulin ( HBB) gene in the germline of human embryos. In this research, six abnormal embryos not suitable for in vitro fertilization were used. The mutation could be corrected in only one of the embryos. Although the mutation could be corrected in two other embryos, nontarget effects occurred in other genes. In the other three embryos, the mutation could not be corrected. It has been reported that this technique is not ready for clinical use because of nontarget effects on different genes (Roh et al., 2018; Carroll, 2019). Modifications that occur in germline cells can be transferred to future generations. Scientists think that they can extract genes that cause diseases in the population using CRISPR-Cas9 (Cai et al., 2018; Memi et al., 2018).

4. Bioethical issues

The fact that CRISPR-Cas9 is among the important discoveries of the 21st century is widely accepted in the scientific community and related industries. However, the rapid rise of CRISPR-Cas9 has led to new bioethical, social, and legal issues in medicine, agriculture, livestock, and the environment. Possible risks and bioethical issues related to CRISPR-Cas9 are summarized in the Table.

Table

Organism

Risks

Bioethical issues

References

Bacteria

Nontarget mutationsGene drifts

Ecological imbalance

Rodriguez, 2016 Hundleby and Harwood, 2019 Esvelt et al., 2014

Plants

Nontarget mutationsGene drifts

Ecological imbalancePatenting

Shinwari et al., 2017 Hundleby and Harwood, 2019

Animals /chimeric animals

Nontarget mutations

Ecological imbalance Patenting Animal welfare and dignity Threatening of human dignity and identity

Rodriguez, 2016 Polcz and Lewis, 2016 Rodriguez, 2017 Eriksson et al., 2018 Koplin, 2019 Degrazia, 2019 de Graeff et al., 2019

Humans

Nontarget mutations Side effects Cost Genetic mosaicism

Eugenics Informed consent Enhancement Accessibility Patenting Safety Incomplete or over legislations

Otieno, 2015 Rodriguez, 2016 Duardo-Sánchez, 2017 Shinwari et al., 2017 Greene and Master, 2018 Sherkow, 2018 Cathomen et al., 2019 Hirsch et al., 2019

4.1. Ecological imbalance

In studies using RNA-targeted gene editing methods based on CRISPR-Cas9, nontarget effects should be examined in depth. Since gene drift will persist in a population, possible off-target mutations will continue in each generation. In addition, the number and effect of mutations may increase as generations progress (Rodriguez, 2016; Hundleby and Harwood, 2019). Another concern is the possibility that genes can be transferred to other species in the environment. Transferring the regulated sequences to other species may result in the transmission of negative characteristics to the associated organisms (Esvelt et al., 2014). The distribution of the properties of the entrained genes among the populations can make control very difficult.

4.2. Regulations for consumers

The use of CRISPR-Cas9 to obtain the desired genetic modifications makes it very difficult to identify and regulate genetically modified organisms (GMOs) in the market after they leave the laboratory. Therefore, regulatory agencies, such as the US Food and Drug Administration (FDA), European Medicines Agency (EMA), and others, should consider whether any GMOs are suitable for consumers. However, it is not known exactly how to evaluate the possibilities of a growing market with CRISPR-Cas9 (Ledford, 2015; Hundleby and Harwood, 2019).

One of the dilemmas of CRISPR-Cas9 that concerns all the humanity is patenting. As is known, transgenic organisms of industrial use and also some human gene sequences for clinical purposes have been patented (Rodriguez, 2016; Sherkow, 2018). As technologies such as CRISPR continue to evolve, patent-related issues in many areas of biotechnology will continue to increase in the upcoming years. Even today, there are many such cases of patenting. The best-known case is the patent right case between Zhang and Doudna and Charpentier for the therapeutic use of CRISPR-Cas9 in human cells. In the case concluded on 2 December 2016, it was decided to grant the patent to Caribou Biosciences, which Doudna was the founder of (Donohoue et al., 2018).

4.3. Genome editing for enhancement

The editing of human germline cells with CRISPR-Cas9, which will be discussed later in more detail, is prohibited for various safety reasons. However, the rate of application of CRISPR-Cas9 to somatic cells is gradually increasing in order to transfer the desired characteristics to our lives. Many phenotypic characteristics have a genetic component independent of the environment. By utilizing this feature, CRISPR-Cas9 can be used to improve the performance of athletes, to prevent violent behavior, or to reduce dependence (Rodriguez, 2016). Although gene therapy is often used to treat patients for their own benefit, the criminal justice system may require repeater or dangerous offenders to correct the genes associated with violence by genome editing technologies in the future. One of the biggest dilemmas here is to obtain informed consent for an underage person if the intervention is made during the development of the zygote. This will give parents or guardians the right to make decisions on behalf of minors for nonhealth reasons. Furthermore, when socially assessed, some genetically improved populations or individuals may have some advantages in comparison to others in terms of various features such as mental and physical capacity (Brokowski, 2018). Therefore, the use of CRISPR-Cas9 in genome enhancement should be seriously discussed both socially and morally.

4.4. Military research

The use of CRISPR technology for military purposes is generally considered within the scope of nontherapeutic enhancement and is covered similarly. From this point of view, related bioethical issues are commonly discussed in terms of concepts of benefit/risk, informed consent, and accessibility (Greene and Master, 2018). A notable bioethical problem is the off-target mutations that have been mentioned in relation to other topics. Off-target mutations can cause many undesirable changes in the genome or even lead to fatal diseases. Current information obtained from studies on off-target mutations caused by CRISPR on the genome is very limited. Therefore, the benefit/risk relationship needs to be evaluated carefully. In addition, the possibility that this technology can be used for the production of new biological weapons is frightening.

Another ambiguous issue that needs to be discussed in military enhancement applications is informed consent. It could be difficult to obtain informed consent forms independently without any interaction among individuals due to military training methods, strict norms, and chains of command. Additionally, some soldiers may have difficulty in understanding the concepts of gene therapy and genome editing, as well as the potential risks and benefits of the applications (Greene and Master, 2018).

One important ethical issue is that the use of such technologies will support ongoing inequalities among military parties (Amoroso and Wenger, 2003). CRISPR is currently an expensive technology. Some developed countries might think of using this technology to further strengthen their defenses and even attack underdeveloped or developing countries. This situation could cause a constant tension, making it difficult to provide an environment of peace and stability worldwide.

4.5. Generation of chimeric animals for organ transplantation

Organ transplantation is the replacement of an organ that cannot function in an individual's body with a healthy organ from a living donor or cadaver. The primary purpose is to save the life of the patient, who is in danger of organ failure, and to increase the lifespan and quality of life (Black et al., 2018). The development of chimeric animals may prevent patients from spending precious time waiting for an appropriate donor.

Bioethical issues in the generation of chimeric animals arise from the fact that chimeras contain human nerve and germ cells (Polcz and Lewis, 2016). The two main issues can be summarized as defining the order of nature and the moral disorders caused by how the organism is treated depending on whether the organism is accepted as human or animal. Some people think that chimeric embryos will affect human dignity and identity because they have the power to develop organisms with human-derived cells and tissues. The others state that chimeric organisms containing human cells cannot turn into humans and therefore will not affect human dignity. They also argue that the human-like features imparted to chimeras will neither affect the biological environment nor the moral status of animals and will never reach human consciousness (Koplin, 2019; Degrazia, 2019).

4.6. Animal welfare and dignity

Animal welfare is another bioethical concern encountered during the application of genome editing technologies on animals. First of all, the possibility of off-target mutations in the genome can lead to diseases or different side effects in animals (Ishii, 2017a; Schultz-Bergin, 2018; de Graeff et al., 2019). Such a situation will adversely affect animal welfare (Rodriguez, 2017).

The second bioethical issue to be discussed could be the concerns about "animal dignity" (Eriksson et al., 2018) and alterations in their natural environments and physiological needs (Manesh et al., 2014). Some studies have stated that the use of animals as objects only serving for humans is not ethically or morally acceptable (Martinelli et al., 2014; Fung and Kerridge, 2016; Greenfield, 2017), and such practices can lead to greater control over humans on animals (Ishii, 2017a; de Graeff et al., 2019). Some others think that animals are not bound by any moral law and therefore there is no need for a discussion regarding animal dignity (Heeger, 2015; Shriver and McConnachie, 2018). Schultz-Bergin (2017) stated that animal rights, welfare, and dignity will not be adversely affected since these animals will occur through genome editing technologies. The existence of contrary opinions on this matter indicates that the mentioned bioethical issues will be on the agenda for a long time.

4.7. CRISPR-Cas9 for human germline

The potential for using CRISPR-Cas9 for genome editing in the human germline has raised serious ethical debates. Until 2015, all therapeutic applications in humans were performed in somatic cells using genome editing technologies. However, in 2015, the editing of the human germline performed by Chinese scientist Huang and his team with CRISPR-Cas9 raised new social, moral, and bioethical issues (Liang et al., 2015; Ormond et al., 2017). Bioethical issues caused by genome editing in the germline can be classified into two main topics depending on the success and failure of genome editing technologies (Ormond et al., 2017; Greely, 2019).

4.7.1. Issues that may occur in the failure of germline genome editing

Some of the ethical dilemmas of genome editing in the germline arise from the fact that changes in the genome can be transferred to the next generations. Therapeutic genome editing in somatic cells generally does not cause significant concerns when assessing the risk/benefit balance and the use of informed consent. The application of CRISPR-Cas9 in the germline is considered more problematic because of the risk of causing various mutations and side effects and transferring undesirable changes to future generations (Cyranoski and Reardon, 2015; Brokowski, 2018; Cai et al., 2018; Halpern et al., 2019). In fact, Huang and his team found that nontarget mutations in the genome occurred and the study was terminated earlier than planned (Liang et al., 2015). Nontarget mutations are unintentional mutations in the genome and may have harmful effects on the organism as these mutations can lead to cell death or transformation (Zhang et al., 2014). Frighteningly, researchers have found that mutations caused by CRISPR-Cas9 in embryos are much more common than in mouse or human adult cells (Cyranoski and Reardon, 2015). In a study performed with human embryos, it was stated that nontarget mutations occur only in the exon regions and therefore the number of mutations may be much higher than expected (Liang et al., 2015). Due to the high risk of nontarget mutations, some scientists argue that genome editing studies in germline cells should be terminated and its future should be discussed (Cyranoski and Reardon, 2015). Some scientists state that newly developed CRISPR-Cas9 could reduce or even prevent the number of nontarget mutations. In this method, the efficiency of CRISPR-Cas9 was increased by using Cas9-regulated human iPSCs in region-specific gene targeting (Yumlu et al., 2019).

Another bioethical dilemma is the cost of germline genome editing. Genome editing is an expensive technology (Wilson and Carroll, 2019). While families in rich countries may have the power to cover this cost, families in developing countries may not. This situation may cause children born in developed countries to have an unfair advantage in terms of various characteristics such as intelligence and physical state compared to children in other countries (Otieno, 2015).

CRISPR-Cas9 is based on the use of nuclease enzymes. The nuclease enzymes used may not be as effective as desired and not be able to cut all copies of the target gene, or the cell may begin to divide before genome editing is completed. As a result, a condition called genetic mosaicism can occur (Lanphier et al., 2015). Genetic mosaicism is the presence of genetically different somatic cell populations in an organism and is often masked. Mosaicism can also lead to major phenotypic changes, the formation of fatal genetic mutations (Capalbo and Rienzi, 2017), and some genetic diseases such as Down, Klinefelter, and Turner Syndromes (Otieno, 2015). Therefore, the nuclease cleavage sites should be exactly confirmed and the possibility of mosaicism should be completely eliminated.

One of the important bioethical issues is side effects in embryos. It is pointed out that the possible side effects cannot be predicted before birth and the consequences are not clearly known (Otieno, 2015; Brokowski, 2018). Controls can only be performed in a small group of cells. This limitation causes the effects of genome editing on embryos to be unknown and unprevented until birth. In fact, it should be considered that it may take years for many potential problems to emerge (Lanphier et al., 2015; Halpern et al., 2019).

4.7.2. Issues that may occur in the successful application of germline genome editing

The first of the bioethical issues of successful germline genome editing is the use for nontherapeutic changes (Lanphier et al., 2015; Greely, 2019). Such uses will lead to new questions about breeding (eugenics) of the human species and its position in the universe (Yang, 2015). In one study, the fur color of rats was successfully changed by genome editing (Yoshimi et al., 2014). It is possible that the skin color of people could be changed in the future. Since the characteristics of individuals can be determined by genome editing rather than blood relations, the possibility that children with similar physical and mental health can be born in the same way should be considered (Ishi, 2015).

The second bioethical issue is what the fate of children born using genome editing will be. From whom or where informed consent will be obtained in the case of undesirable effects on behalf of genome-edited children and whether informed consent will give detailed information are important questions (Beriain and del Cano, 2018; Neuhaus and Zacharias, 2018; Sykora, 2018; Knoppers and Kleiderman, 2019). While clear informed consent can be given for genome-edited somatic cells to be used in clinical trials, it is an enigma to whom and how to give precise information about the potential risks involved in germline editing (Lanphier et al., 2015; Neuhaus and Zacharias, 2018; Knoppers and Kleiderman, 2019).

In December 2015, the International Summit on Human Gene Editing was convened to discuss the social, moral, and bioethical issues caused by genome editing in the human germline. The results of the summit concluded that basic and clinical investigations should be continued in accordance with the appropriate legal and ethical regulations; however, genome editing on gametocytes and embryos that would cause hereditary changes in humans was found to be irresponsible. It was therefore emphasized that the use of CRISPR-Cas9 on the human germline should be postponed until a solution is found for existing bioethical, social, legal, and technical concerns and issues (Baltimore et al., 2015). In addition, it was agreed to establish an international forum where such concerns could be addressed continuously and the studies in different countries could be organized together (Baltimore et al., 2015; Lanphier et al., 2015; Olson, 2015). The NIH announced that genome editing studies in human embryos will not be financially supported (Collins, 2015). In spite of the joint decisions that were made, in February 2016, British scientists were allowed to use CRISPR-Cas9 and similar technologies in human embryos for research purposes only (https://www.bbc.com/news/health-35459054). In March 2017, the US National Academy of Sciences and the American Society of Human Genetics published a position statement stating that they should be aware of the scientific and bioethical issues that can be caused by germline genome editing, but, on the other hand, research should continue (Ormond et al., 2017). As of January 2020, 24 countries have forbidden genome editing in human embryos by law and 9 countries have banned it by guidelines. However, there are countries that do not impose strict prohibitions on germline genome editing (Ishii, 2017b; Lau et al., 2018; Macintosh, 2019).

5. Discussion and future directions

Thanks to its high accuracy, ease of use, and relatively low cost, CRISPR-Cas9 offers a wide range of applications for many people in the medical, agricultural, livestock, and environmental sectors. Furthermore, its precision and accuracy are much higher compared to older technologies such as ZFN and TALEN (Mittal, 2019). The powerful effects of CRISPR-Cas9 have raised many social, moral, and bioethical issues.

Discussions have generally focused on the social, bioethical, and legal consequences of using genome editing technology in human germline cells. Scientists generally agree that CRISPR-Cas9 should be allowed for use in the creation of human disease models, and in understanding the development and molecular mechanisms of diseases; however, it should be prohibited for the purposes of eugenics or enhancement. When ethical issues, safety concerns, and application difficulties are considered together, it is predicted that therapeutic genome editing in human embryos will not be possible in the near future. Thus, the risk of hereditary nontarget genetic mutation is higher than the possible treatment benefits and it affects the principle of intentional harm. Nevertheless, it is clear that scientists will apply CRISPR-Cas9 in germline cells in the future if solutions are found to the issues mentioned here (Duardo-Sánchez, 2017; Hirsch et al., 2019). CRISPR-Cas9 must be fully reliable for therapeutic use in germline cells. Social, legal, and bioethical issues should be discussed in detail once genome editing technologies have reached the permissible level of safety for clinical applications in the prevention of genetic diseases (Rossant, 2018; Cathomen et al., 2019). Subsequently, regulatory laws that may eliminate breaches of germline genome editing will need to be reassessed (Rodriguez, 2016; Duardo-Sánchez, 2017; Cathomen et al., 2019; Macintosh, 2019). The therapeutic use of CRISPR-Cas9 and its rapid rise in the medical field are expected to continue. While studies on the use of CRISPR-Cas9 for clinical purposes are continuing, the necessary legal, social, and ethical legislation should be put into practice as soon as possible and the public conscience should not be ignored.

On the other hand, the potential effects of CRISPR-Cas9 in other areas should not be forgotten. CRISPR-Cas9 is not just about social and bioethical issues related to people. Interactions with other organisms and the environment, such as the consideration of the principle of intentional harm in risk assessment, safety measures to prevent ecological degradation, or potential use in genetic enhancement of animals and agriculture products should also be discussed (Rodriguez, 2016; Hirsch et al., 2019). There are serious concerns about changes in the natural ecosystem that may occur if the GMOs produced with CRISPR-Cas9 are released to the ecosystem in a controlled or uncontrolled manner. Considering the applications of CRISPR-Cas9 that protect mosquitoes from malaria parasites (Gantz et al., 2015) or make female mosquitoes infertile (Hammond et al., 2016), the effect of GM mosquitoes on other organisms with which they are associated in their ecosystems cannot be predicted. It is clear that small-scale research in the laboratory does not fully reflect possible changes in the natural ecosystem (Carroll, 2017). In agriculture, another concern about GMOs produced with CRISPR-Cas9 is whether they will be accepted by the public. GMOs that were produced using different technologies in the past faced harsh public reactions (Carroll, 2017). Furthermore, the fact that GMOs produced with CRISPR-Cas9 are difficult to identify outside the laboratory raises safety concerns (Shinwari et al., 2017). Before the launch of such products, the necessary explanations and declarations should be made by the authorities in a transparent and clear manner in order to prevent misjudgments and questions that may occur in the public, and precautions and arrangements should be established to ensure the safety of the public.

Another issue to consider about CRISPR-Cas9 is patenting. Patenting can considerably limit the application of such technologies. Unilateral patenting can significantly increase the profitability of biotechnology companies, which may lead to a rise of bioethical issues. There is disagreement in the scientific community regarding the patenting or nonpatenting of GMOs to be used specifically for therapeutic purposes (Shinwari et al., 2017; Sherkow, 2018). However, there are some who think that patenting will help to eliminate and regulate the deficiencies in the field (Rodriguez, 2016; Shinwari et al., 2017). It should not be forgotten that the most important of these debates about patenting is commercialization and the release of only reliable products.

In recent years, deals between the scientific community and the pharmaceutical and biotechnology sectors for the therapeutic use of CRISPR-Cas9 have raised public safety concerns (Shinwari et al., 2017; Carroll, 2019) The guidelines and legislations that will regulate the content and application of these deals should be prepared as quickly as possible and shared with the public. Due to the challenges and bioethical issues of CRISPR-Cas9, the scientific community and other interested bioethical, social, legal, and governmental parties should be provided with a detailed guide for future processing and use of this technology (Otieno, 2015; Shinwari et al., 2017; Cathomen et al., 2019). In this way, a long-term policy can be developed that will support the scientific development of CRISPR-Cas9 technology together with the discussion of the possible problems in advance and preparation of solution plans.

References

Gene editing in human embryos. A comment on the ethical issues involved. The Ethics of Reproductive Genetics. Cham. 2018. p. 173.

The human genome editing race: loosening regulatory standards for commercial advantage? Trends in Biotechnology 37 (2. 2019. p. 120. [PubMed]

CRISPR-Cas9 Therapeutics: A Technology Overview 2015.

Statement on NIH Funding of Research Using Gene-Editing Technologies in Human Embryos 2015.

Dignity only for humans? A controversy. The Cambridge Handbook of Human Dignity: Interdisciplinary Perspectives. 2015. p. 541.

International Summit on Human Gene Editing: A Global Discussion 2016.

Germline gene therapy in the era of precise genome editing: how far should we go? In: Soniewicka M (editor. The Ethics of Reproductive Genetics. Cham. 2018. p. 157.

Thinking Towards the Future of CRISPR/Cas9 2015.

Efficient gene editing of human induced pluripotent stem cells using CRISPR/Cas9. CRISPR Gene Editing. 2019. p. 137. [PubMed]

Articles from Turkish Journal of Biology are provided here courtesy of The Scientific and Technological Research Council of Turkey

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Bioethical issues in genome editing by CRISPR-Cas9 technology

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Genome editing technologies have led to fundamental changes in genetic science. Among them, CRISPR-Cas9 technology particularly stands out due to its advantages such as easy handling, high accuracy, and low cost. It has made a quick introduction in fields ...

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Aplicación de CRISPR en Ciencias Médicas

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Aplicación de CRISPR en Ciencias Médicas

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https://link.springer.com/article/10.1007/s11033-021-06479-7

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CRISPR-Cas Genome Editing for Horticultural Crops Improvement: Advantages and Prospects

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Horticultural plants, in particular fruit trees, berry crops, and ornamentals, are valuable objects for studying their genetic and biochemical properties. Along with traditional methods of studying these plants, modern molecular genetic technologies are emerging, in particular genome editing using C…

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El Premio Nobel de Química 2020 fue otorgado a las investigadoras Emmanuelle Charpentier y Jennifer A. Doudna “por el desarrollo de un método para la edición del genoma”, lo que se conoce como la tecnología CRISPR / Cas9.
Emmanuelle Charpentier es actualmente la directora de Instituto Max Planck de Biología de la Infección en Berlín y Jennifer A. Doudna es la principal investigadora en los Institutos Gladstone y profesora en la Universidad de California en San Francisco.

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El hallazgo de las tijeras genéticas permite modificar el ADN de animales, plantas y microorganismos con una precisión extremadamente alta. 

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Esta tecnología ha tenido un impacto revolucionario en la comunidad científica ya que está contribuyendo a nuevas terapias contra el cáncer y apunta a la curación de enfermedades hereditarias en un futuro próximo.

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En el libro digital se presenta una breve descripción de la herramienta CRISPR Cas.

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CRISPRCas - Design & share beautiful engaging content with Visme

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https://encyclopedia.pub/entry/11350

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Genetic Engineering Will Change Everything Forever – CRISPR

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Durante los estudios de Charpentier sobre Streptococcus pyogenes, descubrió una molécula previamente desconocida, el ARNtracr. Su trabajo mostró que el ARNtracr es parte del antiguo sistema inmunológico de las bacterias, CRISPR / Cas, que desarma los virus al escindir su ADN.

Charpentier publicó su descubrimiento en 2011. El mismo año, inició una colaboración con Doudna, una bioquímica experimentada con un vasto conocimiento del ARN. Juntas, lograron recrear las tijeras genéticas de las bacterias en un tubo de ensayo y simplificaron los componentes moleculares de las tijeras para que fueran más fáciles de usar.

En un experimento que hizo época, reprogramaron las tijeras genéticas. En su forma natural, las tijeras reconocen el ADN de los virus, pero Charpentier y Doudna demostraron que podían controlarse para poder cortar cualquier molécula de ADN en un sitio predeterminado. Donde se corta el ADN, es fácil reescribir el código de la vida.

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En el podcast que preparé explico de manera más amplia cuál es el uso actual de CRISPR, sus características y funcionamiento.

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How CRISPR lets us edit our DNA | Jennifer Doudna

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How CRISPR lets us edit our DNA | Jennifer Doudna

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TED talk

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How CRISPR lets us edit our DNA | Jennifer Doudna

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Aplicación de la Herramienta CRISPR en el Control de Plagas

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Aplicación de la Herramienta CRISPR en el Control de Plagas

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by 1,2,† 1,3,† 1,4, 2, 5 4 and 1,3,* , , ,

1. Introduction

Annually, phytophagous insects damage one fifth of the world's total crop yield. Biotic stress affects the food security of any country by compromising the quality and quantity of the crop productivity. The yield loss due to insect infestation has devastating impacts on the society such as, hunger and poverty. The combined impact of the emergence and/or re-emergence of insect pests and rapidly growing human population calls for immediate inventions and the use of rigorous and integrated agricultural practices. The FAO estimated that plant diseases and pests are responsible for a 20-40% reduction in the global crop yields per year [ 1]. Researchers predicted a strong decline in the crop yield in response to climate change and weather pattern variations. Climatic changes might increase the risk that is caused by phytophagous pests, thereby turning them into a more harmful threat to the crops [ 2]. Over the past thousands of years, plant breeding has been exploited for constructing insect resistant crop varieties, however, it is laborious, time-consuming, has a stochastic nature, and the screening process is a very challenging practice [ 3]. Further, the unavailability of a resistance source in the gene pool has restricted the scope of breeding a resistant cultivar [ 4, 5]. Under such a scenario, the use of toxic and cost-intensive agrochemicals appeared to be the only convenient solution for crop protection. Considering the toll these chemicals take on the ecosystem, there was an urge to develop genetically stable and fixed plant types [ 6].

Genome editing (GE) can play a pivotal role as it is a more promising and an environmentally friendly answer that can be used to deal with the situation. It all began with the gene-targeting experiments on the protoplast of Nicotiana tabacum which were performed in 1988 [ 7] and the findings in 1993 which supported that DNA double-strand breaks (DSBs) improved the gene-targeting efficacy [ 8]. Since then, the scientific orientation shifted towards the development of targeted genome editing techniques. The adoption of GE systems provided remarkable results in the field of the genetic improvement of crops. Genetic engineering rationalized the biological research world with the introduction of methods involving in vivo genome editing. The GE technique results in base substitutions and/or insertions/deletions (indels) in the target DNA. It includes several techniques, for instance, the use of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the recently established clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system. In contrast to TALENs and ZFNs, the CRISPR/Cas system is more direct and easier to handle as it requires a single guide RNA (gRNA) for target determination with the Cas9 nuclease [ 8, 9]. In the recent past, the preference has shifted from breeding insect-resistant cultivars to making CRISPR/Cas9-mediated modifications in the agronomic traits or targeted mutagenesis in the insect genome. The constant modifications to gene knockout strategies, transgene integration, nucleotide substitution, transcription regulation, etc., have made the CRISPR/Cas system an easy-to-apply, cost-effective, and a widely used technique for manipulations at the genetic levels [ 10, 11, 12]. Biotic stress resistance is one of the traits that is improved by GE, which makes CRISPR/Cas system highly efficient in enhancing global food security, crop protection, and sustainable agriculture ( Figure 1). On the subject of insects, many research groups have reviewed the application of various GE techniques, with special attention being paid to the CRISPR/Cas9 system in arthropods; in spite of this, no inclusive report is available which covers all of the insect pests. The recent developments in the field of molecular biology and omics approaches presented that there has been a peak in the usage of CRISPR/Cas9 technology for insect pest management, and the data from these reports are not summarized in any previously published reviews. In this study, the authors emphasize and explain the prospects and applications of CRISPR/Cas technology in different insect groups for pest management. The CRISPR/Cas9 system has the potential of providing promising approaches for the control of insect pests. Therefore, summing up the CRISPR/Cas9-based control strategies against insect pests is significant in achieving global goals such as sustainable development.

1.1. CRISPR/Cas-Mediated Genome Editing in Insects

Biotechnology and molecular biology have experienced a great transformation and advancement since the development of the CRISPR/Cas9 gene-editing system in the mammalian cells in the year 2012 [ 13]. With the discovery of the homology-dependent cleavage recombination mechanism, the utility of CRISPR/Cas9 was explored in genome editing, and it emerged as an assuring genome editing tool. The growing utility of gene-editing tools as a site-specific genome-editing approach offers infinite opportunities which may have an impact on important agronomic traits such as resistance to biotic stresses [ 14, 15, 16, 17]. The present study reviews the novel opportunities that CRISPR/Cas9 system offers and how it has attracted all of the attention by offering distinct advantages in the area of insect pest control thus ensuring a good crop yield and food security. A combination of gene editing tools and insect manipulation methods have been already used in Drosophila melanogaster Meigen, several tephritids, and mosquitoes which have answered some basic questions about insect biology. Recently, the technology has been utilized for the development of novel pest control strategies [ 18, 19, 20, 21], and it has been proven to be an efficient approach for pest management [ 22, 23, 24]. The advancements in genome editing methods paved the way for inventive pest control methods by the development of genetically modified insects. The CRISPR/Cas technology is evolving as it is very beneficial for efficient tailoring and gene manipulation. The components of the CRISPR/Cas tool (sgRNA and the Cas9 protein) can be delivered in the target organism in form of plasmid DNA, RNA or a ribonucleo-protein (RNP) complex [ 25]. Some of the phytophagous insect orders that have been explored for pest management using genome editing by the CRISPR/Cas system are reviewed in this article ( Table 1).

1.2. Diptera

Diptera is one of the largest insect orders. The insects of this order are two-winged and are called true flies. Dipterans are abundantly present all around the world, thus, the order is diversified. Their larvae are serious agricultural pests on solanceae, cucurbitaceae and other crop plant families.

1.2.1. Drosophila

As it is the model organism, D. melanogaster encouraged and improved the genome editing technology in insects through use of CRISPR/Cas9 [ 25]. Gratz et al. 2013 were the first to report the CRISPR/Cas9 technology-based deletion of 4.6 kb of chromosomal DNA Drosophila genome. The deletion was performed in yellow locus by using two target sgRNAs and an ssODN (single-stranded oligonucleotide donor) template [ 26]. Further, the possible applications and benefits of CRISPR/Cas9 in the designer flies generation were discussed [ 27]. In 2013, scientists developed a strategy to increase the homologous recombination (HR) frequency by utilizing a reintegration vector [ 28], equated the efficacies of CRISPR/Cas9-mediated and TALEN-based homology-directed repair (HDR), made an easily screen-able platform, and established three different HDR methods for site-specific mutagenesis [ 29].

Two different types of parent flies were developed; one was developed with the Cas9 gene under the germline specific promoter, and another was developed, expressing sgRNA in a constitutive manner. The crossing of these parents produced progenies with a transmitted mutation in the germlines. In a similar way, a gRNA-encoding DNA vector was injected into the Cas9-positive transgenic flies which permitted the knockin or knockout of various genes targets [ 30, 31, 32, 33, 34, 35, 36]. Different approaches have been tried to achieve the induction of HDR in flies for example, a gRNA plasmid and donor repair template were injected into transgenic Cas9 embryos [ 37], while the transgenic embryos containing a sgRNA and Cas9 were injected with a donor template plasmid. All of these three components containing the Cas9 gene, sgRNA and donor repair template in a plasmid form were delivered into non-transgenic flies [ 38, 39].

A comparison was then made among the different approaches of facilitating HDR utilizing the same donor plasmid and gRNA, and it was observed that the use of non-transgenic individuals produced lower frequencies of the knockin ones when they were compared to the transgenic ones [ 40]. A mutagenic chain reaction (MCR) method was developed by Gantz et al. in 2015 that could lead to autocatalytic mutations, and they utilized this process for the conversion of mutations from heterozygous to homozygous forms [ 41]. The CRISPR/Cas9 technique was applied for the introduction of a site-specific mutation in the Dα6 subunit of the nicotinic acetylcholine receptor (nAChR). The resistance level for Spinosad-based insecticide for the flies with a Dα6-null mutation was higher than that of the site-specific mutation, and it was demonstrated that the site-specific mutation is directly related to spinosad resistance [ 42]. Studies have also focused on the altered phenotypes, which were obtained after the knockout of specific genes using the CRISPR/Cas9 system [ 43].

To show the direct relation between the efficiency of the CRISPR/Cas9 system and the concentration of sgRNA, the yellow gene was studied, however, the use of increased sgRNA concentrations reduced the adult survival rate [ 44]. The yellow gene was also targeted by Yu et al. 2013, and a great increase in the editing efficiency was demonstrated [ 45]. Through the CRISPR/Cas9 system, researchers introduced site-specific mutations into the white (w) gene of the D. melanogaster and sex-lethal (Sxl) genes of D. suzukii. The phenotype which was obtained by the mutation in the eyes was produced at a low efficiency, and this might have happened because the sgRNA and Cas9-encoding plasmids DNA were injected into the flies, and here, the plasmid DNA needs to be transcribed in vivo, which is not the case with mRNA. Additionally, the specificity of the Drosophila species and the white gene may have led to this low efficiency. Mutant females of the Sxl gene demonstrated abnormal genitalia and reproductive tissues [ 46]. The CRISPR/Cas9-mediated mutation in the alpha subunit of succinyl-CoA synthetase/ligase (Scsα) in Drosophila revealed that Scsα deficiency displayed developmental delays, increased mortality under starvation conditions and impaired locomotor activity [ 47]. Hence, for energy metabolism in Drosophila, the Scsα gene is vital. Asaoka et al. used CRISPR/Cas9 technology to create flies lacking a linear ubiquitin E3 ligase (LUBEL). When these LUBEL-deficient flies were exposed to heat, defective climbing and a reduced survival rate was observed [ 48].

Further, the utilization of the CRISPR/Cas9 system for vital gene mutation such as the clamp gene, troponin C (TpnC), Alk gene, Sex-lethal (Sxl) and white (w) gene in Drosophila has laid a strong foundation for the development of sustainable pest management strategies [ 49, 50, 51, 52, 53]. The sterile insect technique (SIT) is a pest management approach that includes rearing, sterilizing and releasing the sterilized males of the target insect species. The sterile insect technique is an environmentally friendly and successful strategy for pest control. It is a tool that can be used for elementary research on the reproductive biology of pests. The integration of SIT with CRISPR-Cas genome editing might create optimal SIT strains [ 54, 55]. The site-specific editing of the white gene by directly delivering the purified Cas9 protein in the embryo of the D. suzukii simplified the gene editing. For the generation of heritable genetic modifications, a recombinant Cas9 protein could be a way of choice. Choo et al. in 2017 generated a CRISPR/Cas-mediated series of frameshift mutations, leading to genetic sexing strains in Bactrocera tryoni (Froggatt). The knowledge from this study could be exploited for SIT-based pest control [ 56]. In context to SIT, the system of sperm marking helps to monitor the pest population. In D. suzukii, a sperm-marking transgenic strain was developed by using endogenous promoters of D. suzukii in 2019 [ 57]. The Ds hsp70 promoter is used to derive the expression of Cas9 and a small nuclear RNA gene U6 promoter for the expression of gRNA. In this particular study, the co-injections of the helper plasmid were found to be more effective over the preformed RNPs that had been used previously in HRD-based GE [ 58]. The CRISPR/Cas9-mutated white (w-) gene in D. suzukii caused a copulation failure. It also caused a pigmentation deficiency in the testis sheath, which could be a probable reason for the copulation failure. This approach may, therefore, be capable of being used in pest management [ 57, 59] ( Figure 2 A).

The Dipteran group, Tephritid, comprise destructive pests that are a threat to agricultural crops. Genetic approaches for pest management are very promising methods for tephritid population control. This involves genetic manipulations which enables the insect to transmit lethal traits or renders them sterile. Some examples of genetic pest management in tephritidae are mentioned below.

1.2.2. Anastrepha ludens

Anastrepha ludens, which are also known as the Mexfl (North American Plant Protection Organization's Phytosanitary Alert System) belongs to the family of fruit flies. A. ludens is native to Central America and Mexico and is a chief pest of mango and agriculture in lower Rio Grande Valley, Mexico and Central America [ 60, 61]. The species has a relatively long lifespan in comparison to other fruit fly species [ 62]. This quality is responsible for the aggressive invasion of Anastrepha spp. This genus poses a major threat to the yield of different species of fruit [ 63]. The Anastrepha genus is designated as one of three genera that cause a major risk to American agriculture [ 64]. As per the USDA, the Mexfly can cause an estimated damage of worth $1.44 billion in a 5-year time span. Recently, in 2019, Li. et al. used the CRISPR/Cas9 system for targeting the sex determination gene, As-transformer-2 (Astra-2), through the embryonic injection of the RNP complex. They demonstrated that knocking out of this gene led to sterility in some males and intersexual phenotypes in XX chromosome females. Thus, exploring this sex determining gene (Astra-2) can be useful in pest control management [ 63].

1.2.3. Bactrocera dorsalis

Bactrocera dorsalis is a destructive pest that is found in Asian countries. The white and transformer genes were mutated using the CRISPR/Cas9 system. The sgRNA targeted to the transformer gene, and the Cas9 mRNA were co-injected in the B. dorsalis embryos. The mutations in the transformer gene led to a male-biased sex ratio and as well as this, abnormal outer and interior reproductive organs were formed. These mutations were heritable in the next generation, and thus, this gene can be a suitable target for controlling this pest [ 65].

1.2.4. Ceratitis capitata

The Ceratitis capitata, which is also called as the Mediterranean fruit fly, is an aggressive polyphagous pest that is responsible for a huge amount of economic damage. Initially, the eGFP-to-BFP conversion approach demonstrated that the CRISPR-Cas HDR genome editing technique utilizing short and ssDNA repair templates was highly efficient in C. capitata. Possibly, this efficiency could save resources and time during mutagenic screening while targeting the gene which does not show any phenotypic alterations [ 66]. Meccariello et al. in 2017 targeted the segmentation paired gene (Ccprd) and the eye pigmentation gene white eye (we) in C. capitate using the CRISPR-Cas9 system [ 67].

1.3. Lepidoptera

Lepidoptera is one of the world's most described species and comprises numerous taxa. Lepidopterans are the second most invasive pests of stored crop products, and thus, they have scientific and economic importance. Despite there being great interest in this group, there is unsatisfactory progress in the field of the genetic manipulation of them. Lepidopterans have devastating effects on crop yields. The overuse of chemicals to control the major Lepidopteran insect pests like Spodoptera litura, Spodoptera littoralis, Helicoverpa armigera and Plutella xylostella led to the development of resistance in the insects against the traditionally used pesticides. The two key reasons which explain the failure of the routine use of manipulative genetics in Lepidoptera were resistance for RNAi and sensitivity towards inbreeding [ 68, 69]. Luckily, CRISPR/Cas9-mediated GE provided a heritable and ecofriendly solution for pest management. The GE approaches in some of the insect pests belonging to this order are reviewed in this report [ 70].

1.3.1. Helicoverpa armigera

Insects depend on food for their sterol requirements as they are unable to synthesize it. NPC1b is an insect protein that has been identified as an integral membrane protein. The role of NPC1b in Drosophila melanogaster is an uptake of dietary cholesterol [ 71]. The CRISPR/Cas9-edited NPC1b mutant larvae were used to characterize the function of NPC1b in Helicoverpa armigera. NPC1b is vital for the growth and dietary cholesterol uptake of H. armigera, therefore, a limitation in the dietary uptake of cholesterol inhibits the weight gain and food ingestion of the insect. Thus, NPC1b can be a potential target for pest management. However, the technique might have some off-target effects [ 72]. Wang et al. used the CRISPR/Cas9 system in H. armigera and proved that Cry1Ac resistance relies on HaCad (which is also a key receptor of Cry1Ac). Instead of injecting plasmids coding the Cas9 and sgRNA, the eggs were injected with a mixture of Cas9 mRNA and sgRNA, and this resulted in a highly efficient editing of the HaCad locus. This study led towards the use of Bt toxins [ 73]. The CRISPR/Cas9 system also provided an innovative pest control approach against H. armigera by using the antagonist-mediated optimization of the time of mating which ensured maximum fecundity [ 74].

In H. punctigera and H. armigera, the Bt toxin Cry2Ab resistance was linked with a loss-of-function mutation in the ABC transporter gene (ABCA2). To confirm this co-relation between the Cry2Ab resistance in H. armigera and ABCA2 gene, HaABCA2 knockout strains were developed using the CRISPR/Cas9 technique. The knockout strain obtained a significant level of resistance, and this confirmed the role of HaABCA2 in intermediating the toxicity of Cry2Ab and Cry2Aa against H. armigera [ 75]. In H. armigera, four pigment genes, namely, white, ok, brown and scarlet, were also mutated using the CRISPR/Cas9 system which further led to several physiologically altered phenotypes [ 76]. For a better understanding of insect-insecticide response and the management of agronomic pests, the CRISPR/Cas9-mediated knock-out lines were made for the cluster of nine P450 genes in H. armigera. The study provided the basic information for the critical evaluation and identification of the main players in insecticide metabolism [ 77].

1.3.2. Plutella xylostella, Spodoptera, Dendrolimus punctatus and Cydia pomonella

The extra cellular matrix (ECM) of an insect is made up of chitin and protein. Chitin is a naturally occurring amino-polysaccharide that is synthesized by the Chitin synthase 1 (CHS1) gene-encoded protein. Some common examples of chemicals that directly hinder chitin synthesis are Benzoylureas (BPUs), etoxazole and buprofezin. In the CHS1 gene, SNP was reported at position 1017, in which phenylalanine (F) replaced isoleucine (I), and this change was related to etoxazole resistance [ 78]. Another study on the mutation in the CHS1 gene of Plutella xylostella by Douris et al., in 2016 elaborated that the CRISPR/Cas9 system can convincingly elucidate the molecular mode of action of the CHS1-inhibiting bioactive molecules. The same research group also demonstrated a mutation in the PxCHS1 gene which was responsible for the resistance against benzoylureas (BPUs) in P. xylostella [ 79].

The homeotic gene abdominal-A of the Diamondback Moth, Plutella xylostella (L.) [ 80], and the Fall Armyworm (FAW), Spodoptera frugiperda [ 81], was targeted using the CRISPR/Cas9 system. The double sex (PxDSX) gene of the P. xylostella responsible for sex determination in the insect was targeted using the CRISPR/Cas9 system which led to the altered expression of the sex-biased genes [ 82]. A disruption in the normal functioning of the vital genes by an induced series of deletions and insertions in the genomic locus using the CRISPR/Cas9 system provided the groundwork for the development of new tools for pest management. In a report on Lepidopteran pests, Spodoptera litura CRISPR/Cas9-mediated mutagenesis targeted the Slabd-A gene (S. litura abdominal-A). The Slabd-A gene has a central role in abdominal segmentation and segment identity determination in insects. Thus, Slabd-A is involved in embryonic development, and therefore, targeting this gene for genome manipulation resulted in ectopic pigmentation and anomalous segmentation [ 83]. A study on S. litura demonstrated that SlitBLOS2 gene knockout led to a complete disappearance of the white spots and yellow strips on the larval integument, and thus, this provided a marker gene for functional studies and pest control strategies [ 84].

The most essential odor-binding proteins are PBPs (Pheromone Binding Proteins) [ 85]. To demonstrate the function of the PBP gene in S. litura, the PBP3 gene (SlitPBP3) was mutated using the CRISPR/Cas9 system. The response of the obtained SlitPBP3 mutants was compared with that of the wild-type individuals, and the mutants exhibited a reduced response to sex pheromone components [ 86]. The knockouts of the olfactory receptor co-receptor (Orco) gene of Spodoptera littoralis were acquired using the CRISPR/Cas9 system. These knockouts were not able to respond to sex pheromones and plant odors [ 87]. Recently, a study was conducted which targeted three genes in S. frugiperda. The three target genes included two marker genes tryptophan 2, 3-dioxygenase (TO), the biogenesis of lysosome-related organelles complex 1 subunit 2 (BLOS2), and one developmental gene, E93 (a key ecdysone-induced TF promoting development in adult insects). This study envisioned the method using multiple sgRNA injections, and the results have a possible use in functional gene characterization and high-throughput functional genomics screening for an in-depth understanding of the complex mechanisms regulating the crucial pathways of FAW and other invasive pests. In S. exigua, the functional validation of Seα6-KO (Homozygous strain) was performed using the CRISPR/Cas9 system [ 88].

1.3.3. Dendrolimus punctatus

Dendrolimus punctatus, which is commonly called the Pine Caterpillar Moth, has a devastating effect on the resin production of Southeast Asia and China. The embryo development process is well known to have an association with the Dpwnt-1 gene for early body planning. A CRISPR/Cas9-mediated mutation in the DpWnt-1 gene in D. punctatus greatly altered the gene expression, which led to abnormal growth and deformed organ development. In this study, a mixture of EGFP expressing gene cassette and sgRNA/Cas9 mRNA was used for the embryonic injection [ 89].

1.3.4. Cydia pomonella

The Codling Moth, Cydia pomonella, is a major pome fruit pest. The odorant receptor of C. pomonella (CpomOR1) was targeted using the CRISPR/Cas9 system. The CpomOR1 gene expresses at a high quantity in the antennae which encodes the codlemone receptor (an odorant receptor). In this study, early-stage eggs were injected with sgRNA and Cas9 mRNA. The emerging female with the knockout CpomOR1 gene was allowed to mate with a normal male, and the researchers observed that their fecundity and fertility were affected as they produced non-viable eggs [ 90].

1.3.5. Ostrinia furnacalis (Lepidoptera: Pyralidae)

It is a major agricultural pest in Asia that primarily feeds on corn crops. The CRISPR/Cas9 system was used to understand the function of a developmental gene. The knockout study of the Ago1 gene was performed in O. furnacalis (OfAgo1). In this study, an sgRNA/Cas9 mRNA mix was injected in newly laid eggs. In the hatched larvae, the loss of function of the OfAgo1 gene disrupted the cuticle pigment of the seventh abdominal and third thoracic segments. This finding reveals the role of this gene for cuticle pigmentation [ 91]. Future studies in respect of pest control can be performed by following the insect rearing and transfection methods for this insect.

1.3.6. Agrotis ipsilon

It is also known that the Black Cutworm is a harmful pest that feeds on almost all of the important vegetables and grains. The CRISPR/Cas9 system was used for developing the mutants for the tyrosine hydroxylase (AiTH) gene. A mixture of sgRNA and Ca9 mRNA was microinjected in fresh eggs, and they were incubated until they were ready for hatching. Furthermore, the sgRNA and Ca9 mRNA-injected egg groups were observed by the narrowing of the eggshell that led to a failure in the hatching in most of the eggs. Additionally, if they did hatch, the larva was severely dehydrated and died after one day [ 92].

1.3.7. Hyphantria cunea

Hyphantria cunea (Fall Webworm), Drury, is an invasive pest that feeds on important fruits and crop fields worldwide. Li et al. targeted double sex (Hcdsx) gene using the CRISPR/Cas9 technology, and the mutants were observed to have abnormal external genitalia and incomplete sex reversal phenotypes. This led to a reduced sex-specific fecundity. In the same report, the alternative splicing pattern of the Hcdsx gene was also altered using the CRISPR/Cas9 system, and the alterations in the splicing pattern changed the downstream gene expression of vg1, vg2 (encoding vitellogenin) and pheromone binding protein 1, which in turn led to the development of sex-specific sterile phenotypes in the Hcdsx mutants [ 93].

1.3.8. Mythimna separata

Mythimna separata (Walker) is a phytophagous pest that has been recently targeted for GE for the first time. Tang et al. in 2022 optimized the application of the CRISPR/Cas9 system by targeting the NPC1b gene. The NPC1 family proteins are involved in intestinal absorption and sterol trafficking. The eggs were microinjected with Cas9 protein and sgRNA (RNP complex). The optimized methods from this study can be exploited for the designing of novel pest control strategies [ 94].

1.4. Hemiptera

The order Hemiptera includes insects of the three major suborders Auchenorrhyncha (Spittlebugs, Cicadas, Planthoppers and Leafhoppers), Heteroptera and Sternorrhyncha (Whiteflies and Aphids) which feed almost entirely on plant sap. These insects have adapted to a wide range of diets. Hemiptera is the insect order with the common name of bugs (as flies represent the Diptera). The main features of Hemipteran insects are the sucking and piercing mouthparts. Various control strategies have been applied to control the hemipteran insects. CRISPR/Cas9-mediated genetic alterations have shown remarkable potential in providing a simple and heritable approach in the field of pest management and functional studies of the Hemipteran pests.

1.4.1. Nilaparvata lugen

Nilaparvata lugens which are also known as the Brown Planthopper is a highly destructive insect pest in Asia, and given that it is highly fertile, it causes great agricultural losses by sap sucking rice plants and also by transmitting various viruses. For removing the constraints in the field of functional genomic studies, the CRISPR/Cas9 system was used to target two eye pigmentation genes, namely, the cinnabar gene (Nl-cn) and the white gene (Nl-w) of N. lugens. These genes were knocked out and further validated by an RNAi-based knockdown analysis. This capability of introducing precise genetic alterations provides alternative means for understanding the gene function and establishing new approaches for pest control in this non-model pest [ 95].

1.4.2. Diaphorina citri, Homalodisca vitripennis, Bemisia argentifolii and Bemisia tabaci

Diaphorina citri (Asian citrus psyllid) acts as a vector for Candidatus Liberibacter asiaticus (CLas), a pathogenic bacterium. When the psyllid feeds on the citrus plants, it causes the transmission of a disease that is known as Huanglongbing (HLB). Hunter et al. in 2018 made an effort to improve the delivery of the CRISPR components by the addition of Branched Amphiphilic Peptide Capsules, BAPC. Injecting the gene-editing components near the insect ovaries led to the production of a heritable germline with the edited genome in the subsequent generations. It has bypassed the requirement of microinjecting the eggs [ 96]. Later, in 2019, Hunter et al. utilized this BAPC-assisted-CRISPR-Cas9 method for gene targeting and gene editing in insect nymphs and adult insects (Psyllids-Diaphorina citri; Leafhoppers-Homalodisca vitripennis; Whitefly-Bemisia argentifolii). The two genes that were targeted for the knockouts were the Vermillion, Vm, and the thioredoxin gene, TXT. The knockouts changed their eye color and physiology, respectively. The insects that were selected for this study are a threat to food security as they transmit pathogenic bacteria and viruses to plants worldwide. The idea behind the development of this strategy was to change the vectors into non-vectors. The results were them having a reduced lifespan, slower development, reduced fecundity and it changed their eye phenotype. BAPC-assisted CRISPR delivery reformed the approaches to protect food crops from different pathogens and insect vectors [ 97]. Recently, in 2020, a study was performed on B. tabaci, demonstrating the development of a CRISPR-Cas9 based gene editing technique in which the vitellogenic adult females were injected with the CRISPR/Cas9 components rather than the embryos. Here, the Cas9 protein was fused with an ovary-targeting peptide ligand (''BtKV''), leading to efficient and heritable gene editing in the genome of the offspring. These adult injections were easy to administer and there was no need for specialized tools [ 98] ( Figure 2 C).

1.4.3. Euschistus heros

In Euschistus heros, the Neotropical brown stink bug, three genes yellow (yel), tyrosine hydroxylase (th) and abnormal wing disc (awd) were targeted through RNAi to study the gene function. The awd gene knockdown insects had deformed wings and the th gene targeted insects had a lighter cuticle pigmentation. Thus, the RNAi-mediated targeting revealed that distinct malformed phenotypes are linked to both of the genes, but no distinct phenotype was observed for the yel gene. Further, to understand the function of this gene in this insect, the CRISPR/Cas9-mediated knockout technique was developed. However, still there was no distinct phenotype differences in yel gene mutant insect when they were compared to normal insects. For this study, the yel gene-targeting RNP complex was microinjected in the eggs, and the hatched larvae were observed for the altered phenotype studies [ 99].

1.5. Coleoptera

Coleopteran comprise of the largest order of insecta. In this order, the most common pests of the stored crop products are found. The adult insect of coleoptra have forewings that are modified as hard elytra. They include beetles which inhabit an ample variety of habitats.

1.5.1. Tribolium castaneum

Tribolium castaneum, which is also called the Red Flour Beetle, is a pest of stored grains and other agricultural products. They lower the nutritional value of the crop. Their secretions possess carcinogens like benzoquinone. The availability of the genomic database of T. castaneum [ 100, 101] has upgraded the T. castaneum eradication methods from traditional fumigation to the genome editing-based methods. These new methodologies can expand pest management and diminish the damage that is caused to the environment. The CRISPR/Cas9 system was applied to T. castaneum for the first time [ 102]. In this report, Gilles et al. established that a mutated E-cadherin gene led to dorsal closure defects. Gilles' group also attained a homology-directed knockin using the CRISPR system, which turned out to be very efficient [ 103]. Subsequently, this GE system can be used for various species, providing basic information for launching CRISPR-centered transgenic techniques. This could minimize the development-associated voids between the non-model organisms and the model organisms [ 104].

1.5.2. Leptinotarsa decemlineata

In January 2020, the CRISPR/Cas9 system was used for the first time in the Colorado potato beetle (CPB), Leptinotarsa decemlineata. Initially, the vestigial gene (vest) was functionally characterized in CPB through RNAi, which was followed by the establishment of the CRISPR/Cas9 protocol in CPB for the mutagenesis study. The RNAi-induced phenotype of deformed wings reappeared in the vest gene mutants, and this was also developed using the CRISPR/Cas9 system. In this mutagenesis study, the RNP complex was microinjected in <1 h old eggs, and they were further incubated until they were ready for hatching. Altogether, this report provided an improved environmentally friendly pest management methodology [ 105].

1.6. Orthoptera

Orthoptera possess chewing and biting mouthparts, and they damage the crop by biting off different plant parts. The type of damage that is caused by Orthoptera is the direct damage to the plant, and it is not indirect damage. The damage is visible, and it is similar to the damage that is caused by grubs and caterpillars.

Locusta migratoria

Locusta migratoria is also called the locust, and its breakouts pose an unprecedented threat to agriculture, and thus, to the livelihood and food supply of millions of people. The classical eradication policies to deal with locust breakouts depend on chemical insecticides, which take a toll on the farmers' pockets as well as on the environment. Locusts are considered to be important by scientists for molecular studies also. The developmental synchronization among the migration, social behavior and copulation patterns of the insect act as the foundation in determining the density of the locusts [ 106]. The insects' behavioral responses such as mating, feeding, foraging and spawning are directed by the brain. The pheromones are perceived by the peripheral tissues, processed in the nerve tissue and they are passed on to the sensory organs like the olfactory and others. Finally, the brain receives and integrates these signals, thus, the olfactory plays a vital role in insects. To obtain a better insight on the functional genes of Locusta migratoria, the odorant receptor co-receptor (Orco) gene was targeted by the CRISPR/Cas9 system, and consequently, high mutation rates were achieved. The results suggested novel strategies for locust control, and also, the frame of this study may be used in controlling other insects of the same clade such as crickets [ 107].

1.7. Acarina

The family Acaridae is a pest of cereal products and stored grains, having blunt, toothed chelicerae, enabling them to gouge and scrape the plant material.

Tetranychus urticae

A successful genetic transformation or genome editing is yet to be established for chelicerates. Some tick and mite species within this group are of great economic and agricultural importance. Accessibility to a well-developed genome-editing tool would be a significant improvement in this area. The spider mite Tetranychus urticae is a polyphagous pest. They transmit viruses and travel by water and wind. The insecticides such as pyrimidinamines, quinazolines, pyrazoles and pyridazinones block the quinone binding receptor of mitochondrial respiratory complex I in the T. urticae. These chemicals are categorized as mitochondrial electron transport inhibitors (METIs). In the T. urticae strain which are resistant to these, the METIs were identified with a mutation in the PSST homolog of the complex I [ 108]. It was demonstrated that substitution in the H92R amino acid of the PSST homolog is responsible for their resistance against pyridaben. In 2020, Phytoene desaturase gene mutants of T. urticae were developed through an injection of RNP complex into the ovary of virgin females. The mutation caused albino phenotypes in subsequent progenies. This research provided an impetus for the genetic transformation of chelicerates and paved the way for functional studies using the CRISPR/Cas9 system in T. urticae [ 109].

2. The CRISPR/Cas9 System in Pest Management: Challenges and Future Prospects

In the past decade, the CRISPR/Cas9 system has emerged as an elegant and affordable genetic technique, and it is expected to be extensively applied in pest control applications for crop improvement in the near future [ 88]. Even after the rapid progression in the field of the CRISPR/Cas9-based genome editing methods, researchers are facing challenges such as off-target effects, efficient delivery methods and genome editing efficiency. Despite these challenges, the CRISPR/Cas9 system has the potential for revolutionizing the field of agriculture. Now, there is a need to focus on the improvement of the CRISPR/Cas9-based pest management technologies such as combining the SIT with the CRISPR/Cas9 system, which has great pest control capabilities as it targets the reproductive fitness of them ( Figure 2B). Similarly, the utilization of the CRISPR/Cas9 system for transcription regulation could be a promising technique, but the genetically modified organism in such cases will not be "transgene-free" as it will carry a transgene for dead Cas9 (dCas9) and gRNA expression cassette. Recently, one more novel approach has been explored which involves the transportation of the injected RNP complex which is assisted by the ovary-targeting peptide ligand or BAPC in nymphs and adult flies. Using this method, the CRISPR components can be directly injected near the ovary of an adult female fly, thus making it more convenient when it is compared to the microinjections in the eggs or embryos ( Figure 2 C). This approach has a lot of scope, and this similar concept can be applied in other insect orders for pest management.

3. Discussion

Plant pests are likely to have considerable effects on the crop distribution, density, yield and crop spread as a result of climate change. Pesticides act as a vital tool for pest management and their consumption across the world is reported to be over four million tons per year [ 108]. As per the FAO, crop destruction by insect pests leads to the starvation of millions of people. Over and above one thousand pesticides are existing presently in the market including microbial formulations, chemical ones, semi-chemical ones, herbal ones and others. The persistence of pesticides in the environment poses a worldwide threat to human well-being and ecological systems. Thus, the development of alternative pest management strategies is the need of today. The development of new integrated pest management strategies is required, which have zero side effects on the non-target insect population and lead to effective pest control which further can ensure crop improvement and food safety.

Table 1. CRISPR/Cas9-based genome editing in various insects.

Insect Species

Target Gene

Accession Number

Genetic Trait

Mutation
Type

Delivery of CRISPR Components

Findings

References

Drosophila melanogaster

yellow, rosy

NM_057444.3, NM_079613.3

Pigmentation and Mating

Knockout, Knockin

Plasmid

This was the first report using the CRISPR/Cas9 system to mediate efficient genome engineering in Drosophila.

Gratz et al., 2013

yellow, white

NM_057444.3,
NM_079613.3

Pigmentation and Mating

Knockout

Cas9 mRNA and sgRNA

sgRNA concentration-dependant knockout was shown for yellow gene, and highly efficient and varied genome editing efficiencies were shown by different sgRNAs.

Bassett et al., 2013

Ast, Eh, capa, Ccap, Crz, npf, Mip, mir-219, mir-315, white

NM_001300582.1,
NM_079662.3, NM_079828.3, NM_001275917.2, NM_079626.3, NM_080493.3, NM_140714.4, NR_048289.1, NR_048297.1, X76202.1

Knockout

Plasmid

To obtain a Cas9-sgRNA complex for achieving targeted mutagenesis, two transgene vectors harboring expression cassettes for Cas9 and sgRNA were delivered.

Kondo and Ueda, 2013

rosy, DSH3PX1

NM_079613.3, NM_140091.4

Pigmentation and Mating

Knockout, Knockin

Plasmid

Executed efficient and complex genomic manipulations using CRISPR/Cas9-mediated HDR.

Gratz et al., 2014

ebony, yellow, wingless, wnt

NM_079707.4, NM_057444.3, NM_078778.5

Segmentation

Knockout, Knockin

Plasmid

Different promoters were used to drive sgRNA expression, and based on promoter properties, different patterns of expression were observed.

Port et al., 2014

EGFP, mRFP

Chromogenic fluorophores

Knockout

Plasmid

Induction of mutations by injection of an sgRNA into Vasa-Cas9 transgenic fly embryos.

Sebo et al., 2014

white, piwi

NM_057439.2, NM_001298896.1

Pigmentation and Expression of group of small RNA

Knockout, Knockin

Plasmid

Used Cas9 nickase and sgRNA pairs to prevent off-target effects during the generation of indel mutants.

Ren et al., 2014
[ 110]

NM_143253.2,
NM_057439.2, NM_057444.3

Pigmentation

Knockout, Knockin

Plasmid

CRISPR mediated genome editing was shown in Drosophila.

Xue et al., 2014a

yellow,
notch, bam, nos,ms(3)k81, cid

NM_057444.3, NM_001258581.2, NM_057452.4, NM_057310.4, NM_143253.2, NM_079006.4

Physiology

Knockout

Plasmid

A CRISPR/Cas9-mediated conditional mutagenesis system combined with tissue-specific expression of Cas9 was used to temporally and spatially inhibit gene expression.

Xue et al., 2014b

NM_079707.4, NM_057444.3, NM_078558.3

Pigmentation

Knockout, Knockin

Plasmid, transgene

Donor template and sgRNA plasmids were injected into Cas9 transgenic embryos in Drosophila.

Ren et al., 2014b

NM_079707.4, NM_057444.3, NM_057439.2

Pigmentation

Knockout, Knockin

Plasmid, transgene

A bicistronic Cas9/sgRNA vector was constructed which enhanced the efficiency of gene targeting.

Gokcezade et al., 2014

ebony, yellow, wg, wls, Lis1, Se

NM_079707.4, NM_057444.3, NM_078778.5, NM_140188.4, NM_057812.5, NM_139978.4, NM_057812.5

Pigmentation and Physiology

Knockout, Knockin

Plasmid, transgene

Non-transgenic individuals exhibited lessefficient knockin than transgenic individuals did.

Port et al., 2015
[ 111]

NM_135273.5,NM_141949.4

Knockin

mRNA

HDR-mediated genome modifications efficiency was tested, and a problem associated with "ends-in" recombination was resolved.

Yu et al., 2014

fdl

NM_165908.2

Knockout

Plasmid

Capability of CRISPR/Cas9 system for analysing or manipulating protein glycosylation pathways.

Mabashi-Asazuma et al., 2015
[ 112]

mod(mdg4)

NM_163878.2

Knockout

Plasmid

Validation of a functional gene involved in trans-splicing that influenced the development in flies.

Gao et al., 2015
[ 113]

NM_167053.2, NM_132679.3, L76559.1

Knockout, Knockin

Plasmid, transgene

Offered a comprehensive technique for genome editing in Drosophila S2 cells.

Kunzelmann et al., 2016
[ 114]

NM_057444.3, NM_057439.2,
NM_132315.1

Pigmentation

Knockin

Plasmid, transgene

Proposed a new process of attaining single or multiple allelic substitutions.

Lamb et al., 2016

white, Sxl

NM_057439.2, XM_017083263.2

Sex determination

Knockout

Plasmid

Sxl gene was proved as excellent gene to suppress the population growth of this destructive pest.

Li and Scott, 2016

DsRed
(red fluorescence protein)

knockin

Plasmid, transgene

The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used for expression of fluorescent proteins or effector molecules in testes of pests, and this providing basis for reproductive biology studies sexing and monitoring.

Ahmed, H. M. et al., 2019

Drosophila subobscura

yellow, white

XM_034814491.1,
XM_034808177.1

Pigmentation

Knockout

mRNA

Gene functions were analyzed in a non-model Drosophila species.

Tanaka et al., 2016
[ 115]

Bactrocera dorsalis

White and transformer

AY055817.1, KP342062.1

Sex determination and reproduction

Knockout

RNP complex

CRISPR/Cas9 mediated mutation of white and transformer genes caused various phenotypic effects.

Zhao et al., 2018

Ceratitis capitata

eGFP_gRNA2b-Cas9 complexes with ssODN_BFP donor template

Homology directed repair

knockin

RNP complex and a single-stranded oligo donor

Conversion of eGFP-to-BFP was demonstrated for establishing an efficient HDR through CRISPR-based genome editing.

Aumann, R. A. et al., 2018

X89933.1, XM_020858622.2

Segmentation

Knockout

RNP complex

A simple and highly efficient RNP complex-based genome editing approach was reported with the details of designing and preparation.

Meccariello, A. et al., 2017

HaABCA2

KP259911.1

Regulation of enzymes

Knockout

Cas9 mRNA and sgRNA

Wang, J. et al., 2017

odorant
receptor 16 (OR16)

KF768670.1

Olfaction

Knockout

Cas9 mRNA + sgRNA and RNP complex

The results represent the basis for novel olfactory-based strategies of pest population
control.

Chang, H. et al., 2017

white, ok, brown, and scarlet

XM_021344759.2, KU754490.1, KU754480.1, KU754478.1

Pigmentation

Knockout

Cas9 mRNA

The report represented differential distribution of eye pigments in the mutants; this finding may be helpful in elucidation of biosynthetic pathway.

Khan, S. A.,et al., 2017

cluster of nine P450 genes

KM016735.1, R095600.1, KM016739.1, KM016740.1, DQ256407.1, KM016743.1, KM016741

Regulation of detoxifying enzymes

Knockout

Cas9 protein and multiple sgRNAs

The report identified the key players in the insecticide metabolism.

Wang, H. et al., 2018

Pxdsx

XM_048630440.1

Sex determination

Knockout

Microinjection of RNP complex

The results showed CRISPR/Cas9 system led to altered expression of sex biased genes.

Wang et al., 2019

PxCHS1

AB271784.1

Development

Knockout

Plasmid

Description of the resistance management strategies for insect pests, it and explained the MoA behind the resistance using CRISPR/Cas9 system.

Douris et al., 2016

LW

Locomotion

Knockout

RNP complex

The results showed weaker phototaxis and reduced locomotion, thus making it a helpful method for pest control

Chen et al., 2021
[ 116]

XM_035582273.2
XM_050696092.1
XM_050696079.1

Growth and development

Knockout

Cas9 protein and multiple sgRNAs

The developed mutants were helpful to understand the crucial pathways of S. frugiperda and the strategy can also applied for other invasive pests.

Zhu, G. H. et al., 2020
[ 117]

Spodoptera litura

Slabd-A

GCA_002706865.1

Body segmentation

Knockout

Cas9 mRNA and sgRNA

Bi, H. L. et al., 2016

Spodoptera littoralis

SlitOrco

Olfaction

Knockout

mRNA

The Orco gene was investigated in the insect Spodoptera littoralis. The results were helpful in making a pest control strategy and in gene function analysis.

Koutroumpa et al., 2016

Hyphantria cunea

Hcdsx

Reproduction

Knockout

sgRNA and Cas9 mRNA

Knocked-out Hcdsx gene by CRISPR/Cas9 caused sex-specific sterility, thus making it a pest control method.

Li et al., 2020

Nilaparvata Lugens

Nl-cn and
Nl-w

MH105806.1

Pigmentation

Knockout

Cas9 mRNA and sgRNA

Two genes for eye pigmentation were targeted using CRISPR/Cas9, and the results paved path for gene-function interrogation.

Xue. et al., 2018

Diaphorina citriHomalodisca vitripennis, Bemisia argentifolii

Thioredoxin and Vermillion

XM_046819472.1

Physiology andEye color

Knockout

BAPC, plasmid, dsRNA

The BAPC-assisted delivery system developed gene editing methods across the all hemipteran pests by permitting the use of nymphs and adults. BAPC-assisted CRISPR delivery transformed the approaches to protect food crops from different pathogens and insect vectors.

Hunteret al., 2019

Euschistus heros

abnormal wing disc (awd), tyrosine hydroxylase (th) and yellow (yel)

NP_001119625.1, XP_008182999.1, XP_001948479.1

Body segmentation and pattern

Knockdown and knockout

dsRNA, RNP complex

Use of RNAi and CRISPR/Cas9 techniques for managing insect pests.

Cagliari et al., 2020

Author Contributions

S.S., S.R., S.P., G.S., S.K.U., G.M. and P.C.V. have conceived and planned this article and all the authors have reviewed it. S.S. and S.R. wrote the first draft; all authors contributed to and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by SERB Department of Science and Technology (Govt. of India) funded project under the grand agreement CRG2020001095.

Institutional Review Board Statement

Approved for publication. Institutional Manuscript ID no. CSIR-NBRI_MS/2022/07/14.

Informed Consent Statement

Data Availability Statement

Acknowledgments

P.C.V. and G.M. are thankful to the SERB, Department of Science & Technology (DST), Govt. of India for the financial support (CRG/2020/001095). The authors would like to thank CSIR, National Botanical Research Institute, Lucknow (U.P.), India for necessary support and facilities.

Conflicts of Interest

Authors have no conflict of interest to declare.

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Figure 1. The figure shows various genes targeted by CRISPR/Cas9 for insect pests control.

Figure 2. Figure shows three different CRISPR-based genome-editing approaches for insect pests management. ( A) pictorial representation of sterile insect technique (SIT); ( B) targeting female reproductive fitness through CRISPR/Cas9; ( C) injection of CRISPR/Cas9 components with ovary targeting signals for producing heritable genome editing in the germline.

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Singh, S.; Rahangdale, S.; Pandita, S.; Saxena, G.; Upadhyay, S.K.; Mishra, G.; Verma, P.C. CRISPR/Cas9 for Insect Pests Management: A Comprehensive Review of Advances and Applications. Agriculture 2022, 12, 1896. https://doi.org/10.3390/agriculture12111896

AMA Style

Singh S, Rahangdale S, Pandita S, Saxena G, Upadhyay SK, Mishra G, Verma PC. CRISPR/Cas9 for Insect Pests Management: A Comprehensive Review of Advances and Applications. Agriculture. 2022; 12(11):1896. https://doi.org/10.3390/agriculture12111896

Chicago/Turabian Style

Singh, Sanchita, Somnath Rahangdale, Shivali Pandita, Gauri Saxena, Santosh Kumar Upadhyay, Geetanjali Mishra, and Praveen C. Verma. 2022. "CRISPR/Cas9 for Insect Pests Management: A Comprehensive Review of Advances and Applications" Agriculture 12, no. 11: 1896. https://doi.org/10.3390/agriculture12111896

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CRISPR/Cas9 for Insect Pests Management: A Comprehensive Review of Advances and Applications

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Insect pests impose a serious threat to agricultural productivity. Initially, for pest management, several breeding approaches were applied which have now been gradually replaced by genome editing (GE) strategies as they are more efficient and less laborious.

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¿Qué es la tecnología CRISPR/Cas9 y cómo nos cambiará la vida?

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¿Qué es la tecnología CRISPR/Cas9 y cómo nos cambiará la vida?

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Por: Alberto Morán

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Las posibilidades son casi inimaginables. Con la tecnología CRISPR/Cas9 se inaugura una nueva era de ingeniería genética en la que se puede editar, corregir, alterar, el genoma de cualquier célula de una manera fácil, rápida, barata y, sobre todo, altamente precisa. Cambiar el genoma significa cambiar lo esencial de un ser, recordadlo.

En un futuro relativamente cercano servirá para curar enfermedades cuya causa genética se conozca y que hasta ahora eran incurables. Es lo que seguramente muchas veces habréis oído nombrar como terapia génica. Ya se está trabajando con esta tecnología en estas enfermedades como la Corea de Huntington, el Síndrome de Down o la anemia falciforme. Otra aplicación aparentemente futurista, pero no tan quimérica es la reprogramación de nuestras células para que corten el genoma del VIH.

Así, el MIT (Instituto Tecnológico de Massachussets) anunció ya en marzo de 2014 que había conseguido curar a un ratón adulto de una enfermedad hepática ( tirosinemia de tipo I) de origen genético utilizando esta tecnología.

Y, sí, como muchos estáis pensando, esta técnica también vale para modificar los genomas de embriones humanos. Sobre las implicaciones éticas y sociales habría que escribir muchos libros, pero no es nuestro objeto aquí.

Además de estas implicaciones sanitarias, también se puede utilizar para mejorar los alimentos transgénicos (desarrollar nuevas variedades de plantas y animales con características genéticas concretas), modificar bacterias y otros microorganismos de uso industrial y alimentario.
Guerra de patentes
La ciencia también tiene una parte "fea". Y en esta ocasión, como casi siempre, viene derivada de los intereses económicos (obvios) que tiene la tecnología CRISPR/Cas9.

Poco después del famoso artículo de Doudna y Charpentier, en enero de 2013 los laboratorios de George Church en Harvard y Feng Zhang en el Broad Institute del MIT fueron los primeros en publicar artículos demostrando que CRISPR/Cas9 servía para células humanas. Doudna publicó lo propio de manera independiente apenas unas semanas más tarde.

En abril de 2014 Zhang y el Instituto Broad obtuvieron la primera de entre varias patentes generales que cubren el uso de CRISPR en eucariotas. Eso les otorgaba los derechos para usar CRISPR en ratones, cerdos, humanos... prácticamente en cualquier criatura que no fuera una bacteria.

La velocidad de obtención de la patente sorprendió a algunos. Y fue porque el Instituto Broad había pagado de una manera discreta para que la revisaran muy rápido, en menos de seis meses. Además el proceso se llevó a cabo de una manera casi "secreta". Junto con la patente llegaron más de mil páginas de documentos. Doudna había presentado antes que la de Zhang, una solicitud de patente. Pero, según Zhang, la predicción de Doudna en su solicitud de que su descubrimiento funcionaría en humanos era una "mera conjetura" y que en cambio él fue el primero en demostrarlo en un acto de invención distinto y "sorprendente". Para demostrar que fue "el primero en inventar" el uso de CRISPR-Cas en células humanas, Zhang presentó fotos de cuadernos de laboratorio que según él demuestran que tenía el sistema en marcha a principios de 2012, incluso antes de que Doudna y Charpentier publicaran sus resultados o solicitaran su propia patente. Esa cronología significaría que él descubrió el sistema CRISPR-Cas independientemente. En una entrevista, Zhang afirmó que había hecho los descubrimientos él solo. Al preguntársele qué había aprendido del artículo de Doudna y Charpentier, dijo "no mucho".

Por otra parte, los abogados de Doudna y Charpentier no se van a quedar de brazos cruzados y se espera que monten un "procedimiento de interferencia" en Estados Unidos, que es un proceso legal en el que el ganador se lo lleva todo y en el que un inventor puede hacerse con la patente de otro.

En el juego de las patentes aparecen implicadas tres start-up para las que el control de esas patentes es clave. Entre esas empresas están Editas Medicine, Intellia Therapeutics, ambas de Cambridge, y CRISPR Therapeutics, una start-up de Basilea (Suiza) cofundada por Charpentier. Zhang cofundó Editas Medicine, que en diciembre de 2014 anunció que había comprado la licencia para el uso de su patente al Instituto Broad. Pero Editas no tiene el monopolio sobre CRISPR porque Doudna también fue cofundadora de la empresa. Y desde que salió la patente de Zhang, Doudna ha roto con la empresa, y su propiedad intelectual en forma de su propia patente pendiente de aprobación se ha vendido bajo licencia a Intellia. Para complicar aún más las cosas, Charpentier vendió sus derechos de la misma solicitud de patente a CRISPR Therapeutics.

Por otra parte, cada vez hay más voces que piden que, debido a la gran capacidad de curar enfermedades por parte de CRISPR-Cas9, la tecnología no quede protegida por patente y se deje como acceso público. La propia Charpentier asegura que la tecnología ha sido puesta libremente a disposición de la comunidad investigadora, por lo que no cree que la patente suponga ningún obstáculo al avance científico.

Veremos qué nos depara el futuro.

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¿Qué es la tecnología CRISPR/Cas9 y cómo nos cambiará la vida?

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ESTA INFORMACIÓN HA SIDO ACTUALIZADA EN ESTE ARTÍCULO. La tecnología CRISPR/Cas9 es una herramienta molecular utilizada para "editar" o "corregir" el genoma de cualquier célula. Eso incluye, claro está, a las células humanas. Sería algo así como unas tijeras moleculares que son

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En 2005, Francisco J. Martínez Mojica publicó el descubrimiento que desató la revolución CRISPR.
Martínez Mojica identificó la secuencia CRISPR en microorganismos y descubrió que se trataba de un sistema de defensa contra los virus; Charpentier y Doudna elucidaron el mecanismo molecular de CRISPR/Cas 9 y demostraron que tenía un uso potencial como herramienta universal de edición genómica.