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CRISPR refere-se a "repetições palindrómicas curtas agrupadas e regularmente intercaladas".

Cas9 é uma enzima que pode cortar DNA.

Em conjunto, podem ser programados para alvejar sequências específicas de DNA e introduzir cortes precisos, que podem então ser reparados de maneira desordenada (causando uma mutação) ou usando um modelo para introduzir uma sequência desejada.

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Joung, J. K., & Sander, J. D. (2013). TALENs: a widely applicable technology for targeted genome editing. Nature Reviews Molecular Cell Biology, 14(1), 49-55.

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TALENs: a widely applicable technology for targeted genome editing - Nature Reviews Molecular Cell Biology

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Transcription activator-like effector nucleases (TALENs) comprise a nonspecific nuclease fused to a sequence-specific DNA-binding domain. This domain can be engineered so that TALENs can target virtually any sequence. TALENs are an efficient tool to modify genes in a wide range of cell types and organisms.

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Convenção sobre Direitos Humanos e Biomedicina (Oviedo, 1997): Proíbe a manipulação genética que vise a modificação da linha germinal humana.

Declaração Universal sobre o Genoma Humano e Direitos Humanos (UNESCO, 1997): Enfatiza o respeito pela dignidade humana no contexto da genómica.

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Consiste em fazer alterações precisas em sequências específicas de DNA em células vivas.

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Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., & Gregory, P. D. (2010). Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 11(9), 636-646.

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Key Points

Targeted genetic engineering in many important model systems and in human tissue culture cells has historically been challenging. This has changed dramatically over the past 5 years with the development of zinc finger nuclease (ZFN) technology.

A ZFN is an artificial endonuclease that consists of a designed zinc finger protein (ZFP) fused to the cleavage domain of the Fok I restriction enzyme. A ZFN may be redesigned to cleave new targets by developing ZFPs with new sequence specificities.

For genome engineering, a ZFN is targeted to cleave a chosen genomic sequence. The cleavage event induced by the ZFN provokes cellular repair processes that in turn mediate efficient modification of the targeted locus.

If the ZFN-induced cleavage event is resolved via non-homologous end joining, this can result in small deletions or insertions, effectively leading to gene knockout. This approach has now been used to establish facile and efficient reverse genetics (that is, reverse genetics that does not require selection) in Drosophila melanogaster, zebrafish, rats, Arabidopsis thaliana and mammalian somatic cells.

If the break is resolved via a homology-based process in the presence of an investigator-provided donor, small changes or entire transgenes can be transferred, often without selection, into the chromosome; this is referred to as 'gene correction' and 'gene addition', respectively. This approach has been used to make novel alleles in D. melanogaster, mammalian cells and tobacco, and has been used to drive targeted integration in maize, tobacco and human embryonic stem and induced pluripotent stem cells.

Therapeutic application of ZFN technology requires the engineering of ZFNs that are highly specific in their action. Three clinical trials with ZFNs are underway, including one in which T cells are isolated from a patient infected with HIV, treated with ZFNs that disrupt the chemokine (C-C motif) receptor type 5 ( CCR5) gene to make them resistant to virus infection, and transferred back to the patient.

Abstract

Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells - this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.

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References

1. Bibikova, M., Beumer, K., Trautman, J. K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003). References 2 and 3 are early studies in the field of genome editing that applied ZFNs for the disruption and correction of endogenous genes in D. melanogaster.

2. Miller, J., McLachlan, A. D. & Klug, A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 4, 1609-1614 (1985). This paper describes the discovery of the Cys2-His2 zinc finger - the most common DNA recognition motif in metazoa.

3. Pavletich, N. P. & Pabo, C. O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science 252, 809-817 (1991). This study describes the first X-ray structure of a Cys2-His2 ZFP-DNA complex.

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7. Rouet, P., Smih, F. & Jasin, M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell Biol. 14, 8096-8106 (1994). The conclusion drawn by this paper - that the DNA ends generated by a DSB in a mitotically dividing mammalian cell are recombinogenic - provided a starting point for the development of genome editing. See also reference 57.

8. Bozas, A., Beumer, K. J., Trautman, J. K. & Carroll, D. Genetic analysis of zinc-finger nuclease-induced gene targeting in Drosophila. Genetics 182, 641-651 (2009). This paper provides the first comprehensive study of the intranuclear requirements for resolving an endogenous DSB by NHEJ rather than HDR.

9. DeKelver, R. C. et al. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human. Genome Res. 20, 1133-1142 (2010). A useful feature of ZFN-driven genome editing is the ability it provides to perform mammalian somatic cell genetics experiments in isogenic settings. This paper provides several examples of such isogenic engineering.

10. Perez, E. E. et al. Establishment of HIV-1 resistance in CD4⁺ T cells by genome editing using zinc-finger nucleases. Nature Biotech. 26, 808-816 (2008). This paper provides data underlying the first clinical trial on the genome editing of human cells. See also reference 81.

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14. Rebar, E. J. & Miller, J. C. Design and applications of engineered zinc finger proteins. Biotech International 20-23 (April/May 2004).

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Authors and Affiliations

Sangamo BioSciences Inc., 501 Canal Boulevard, Suite A100, Richmond, 94804, California, USA

Fyodor D. Urnov, Edward J. Rebar, Michael C. Holmes, H. Steve Zhang & Philip D. Gregory

Corresponding author

Correspondence to Philip D. Gregory.

Ethics declarations

Competing interests

Fyodor D. Urnov, Edward J. Rebar, Michael C. Holmes, H. Steve Zhang and Philip D. Gregory are full time employees of Sangamo BioSciences Inc.

Glossary

Paralogues

Two or more genes within a given species that originated from a single parent gene via duplication events, usually with a subsequent, sometimes subtle, divergence of function.

Pseudogenes

Non-functional paralogues that often lack promoter or intron sequences.

Adeno-associated viruses

(AAVs). As defined by Flint and colleagues in Principles of Virology, AAV is a parvovirus (also known as a dependovirus) that can establish a latent infection during which its DNA is integrated into the host cell genome in an inactive state. AAV2 integrates into the PPP1R12C gene locus on chromosome 19.

Homing endonuclease

A 'meganuclease', such as I- SceI, that recognizes and cuts longer sequences (18 bp in the case of I- Sce I) than those cut by commonly used restriction enzymes.

Episomal

The formal, technical definition of 'episome', and its distinction from the word 'plasmid' (as proposed by Jacob and Wollman in 1958), is complex. In vernacular use it means 'circular extrachromosomal DNA molecule present inside the nucleus'.

Protoplasts

Plant cells that lack a cell wall.

Calli

(Sing. callus.) Clusters of undifferentiated plant cells grown on solid medium that, in some species and under specific culture conditions, have the capacity to regenerate a whole plant.

Short hairpin RNA

Small RNAs that form hairpins that can induce sequence-specific silencing in mammalian cells through RNAi.

Systematic evolution of ligands by exponential enrichment

(SELEX). A method for identifying nucleic acid ligands for a chosen 'bait' molecule (typically a protein). In its most general form, the method comprises: incubation of a randomized nucleic acid library with the bait molecule; recovery of the bait, along with any bound nucleic acids; amplification of recovered nucleic acids via PCR; and sequencing to identify binding motifs.

Embryogenic suspension cells

Plant cells, derived from callus tissue or from an embryo, that can be maintained in liquid growth medium and that, under appropriate culture conditions, can be used to regenerate a whole plant.

Autologous

In transplantology, referring to cells or an organ transplanted from an individual to that same individual (often after some ex vivo procedure has been performed).

Intrabodies

Antibodies that are directed against intracellular target molecules and expressed within a specific subcellular compartment as directed by localization signals genetically fused to the amino or carboxyl terminus of a given antibody.

Aviraemic

Aviraemia refers to the lack of detectable virus in the circulation of an individual.

Ultradeep sequencing

An umbrella term that refers to several independent, proprietary, high-throughput DNA-sequencing technologies that use massively parallel sequencing-by-synthesis approaches. The new methods allow an increase in generated sequence per run of about two orders of magnitude compared with conventional Sanger sequencing technologies.

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Urnov, F., Rebar, E., Holmes, M. et al. Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11, 636-646 (2010). https://doi.org/10.1038/nrg2842
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# data[].attributes.attachment_link.title
Genome editing with engineered zinc finger nucleases - Nature Reviews Genetics

# data[].attributes.attachment_link.description
Zinc finger nucleases (ZFNs) are versatile tools for making precise modifications to genomes, and their use is now established in a range of model systems. ZFNs are also showing potential in human gene therapy, and several clinical trials are underway.

# data[].attributes.body
As TALENs são proteínas artificiais que podem ser projetadas para se ligarem a locais específicos no DNA.

Ao serem fusionadas com uma enzima de corte de DNA, elas podem introduzir cortes em locais específicos, permitindo a edição de genes.

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Semelhante às TALENs.

ZFNs são proteínas que podem ser projetadas para reconhecer e se ligar a sequências específicas de DNA.

Elas também são fusionadas com uma enzima de corte para permitir a edição de genes.

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Recombinase mediada por troca de cassete (RMCE)

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Recombinase mediada por troca de cassete (RMCE)

# data[].attributes.body
Esta é uma técnica que utiliza enzimas recombinases para trocar sequências específicas de DNA por outras.

É útil para introduzir sequências exógenas em locais específicos de um genoma.

# data[].attributes.body
Este é um processo natural pelo qual as células reparam quebras de DNA usando uma sequência homóloga como modelo.

Em edição de genes, os investigadores podem fornecer um modelo de DNA que contém as mudanças desejadas, e a célula usará esse modelo para reparar o corte, resultando na introdução das mudanças desejadas.

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Uma das aplicações mais esperadas é a correção de mutações genéticas causadoras de doenças.

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A edição de genes pode ser utilizada para desenvolver culturas mais resistentes e nutritivas.

# data[].attributes.body
Organismos geneticamente modificados podem ser utilizados para produzir medicamentos.

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Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262-1278.

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Abstract

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

Introduction

The development of recombinant DNA technology in the 1970s marked the beginning of a new era for biology. For the first time, molecular biologists gained the ability to manipulate DNA molecules, making it possible to study genes and harness them to develop novel medicine and biotechnology. Recent advances in genome engineering technologies are sparking a new revolution in biological research. Rather than studying DNA taken out of the context of the genome, researchers can now directly edit or modulate the function of DNA sequences in their endogenous context in virtually any organism of choice, enabling them to elucidate the functional organization of the genome at the systems level, as well as identify causal genetic variations.

Broadly speaking, genome engineering refers to the process of making targeted modifications to the genome, its contexts (e.g., epigenetic marks), or its outputs (e.g., transcripts). The ability to do so easily and efficiently in eukaryotic and especially mammalian cells holds immense promise to transform basic science, biotechnology, and medicine ( Figure 1 ).

For life sciences research, technologies that can delete, insert, and modify the DNA sequences of cells or organisms enable dissecting the function of specific genes and regulatory elements. Multiplexed editing could further allow the interrogation of gene or protein networks at a larger scale. Similarly, manipulating transcriptional regulation or chromatin states at particular loci can reveal how genetic material is organized and utilized within a cell, illuminating relationships between the architecture of the genome and its functions. In biotechnology, precise manipulation of genetic building blocks and regulatory machinery also facilitates the reverse engineering or reconstruction of useful biological systems, for example, by enhancing biofuel production pathways in industrially relevant organisms or by creating infection-resistant crops. Additionally, genome engineering is stimulating a new generation of drug development processes and medical therapeutics. Perturbation of multiple genes simultaneously could model the additive effects that underlie complex polygenic disorders, leading to new drug targets, while genome editing could directly correct harmful mutations in the context of human gene therapy ( Tebas et al., 2014).

Eukaryotic genomes contain billions of DNA bases and are difficult to manipulate. One of the breakthroughs in genome manipulation has been the development of gene targeting by homologous recombination (HR), which integrates exogenous repair templates that contain sequence homology to the donor site ( Figure 2A ) ( Capecchi, 1989). HR-mediated targeting has facilitated the generation of knockin and knockout animal models via manipulation of germline competent stem cells, dramatically advancing many areas of biological research. However, although HR-mediated gene targeting produces highly precise alterations, the desired recombination events occur extremely infrequently (1 in 10 ⁶-10 ⁹ cells) ( Capecchi, 1989), presenting enormous challenges for large-scale applications of gene-targeting experiments.

To overcome these challenges, a series of programmable nuclease-based genome editing technologies have been developed in recent years, enabling targeted and efficient modification of a variety of eukaryotic and particularly mammalian species. Of the current generation of genome editing technologies, the most rapidly developing is the class of RNA-guided endonucleases known as Cas9 from the microbial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats), which can be easily targeted to virtually any genomic location of choice by a short RNA guide. Here, we review the development and applications of the CRISPR-associated endonuclease Cas9 as a platform technology for achieving targeted perturbation of endogenous genomic elements and also discuss challenges and future avenues for innovation.

Programmable Nucleases as Tools for Efficient and Precise Genome Editing

A series of studies by Haber and Jasin ( Rudin et al., 1989; Plessis et al., 1992; Rouet et al., 1994; Choulika et al., 1995; Bibikova et al., 2001; Bibikova et al., 2003) led to the realization that targeted DNA double-strand breaks (DSBs) could greatly stimulate genome editing through HR-mediated recombination events. Subsequently, Carroll and Chandrasegaran demonstrated the potential of designer nucleases based on zinc finger proteins for efficient, locus-specific HR ( Bibikova et al., 2001, 2003). Moreover, it was shown in the absence of an exogenous homology repair template that localized DSBs can induce insertions or deletion mutations (indels) via the error-prone nonhomologous end-joining (NHEJ) repair pathway ( Figure 2A ) ( Bibikova et al., 2002). These early genome editing studies established DSB-induced HR and NHEJ as powerful pathways for the versatile and precise modification of eukaryotic genomes.

To achieve effective genome editing via introduction of site-specific DNA DSBs, four major classes of customizable DNA-binding proteins have been engineered so far: meganucleases derived from microbial mobile genetic elements ( Smith et al., 2006), zinc finger (ZF) nucleases based on eukaryotic transcription factors ( Urnov et al., 2005; Miller et al., 2007), transcription activator-like effectors (TALEs) from Xanthomonas bacteria ( Christian et al., 2010; Miller et al., 2011; Boch et al., 2009; Moscou and Bogdanove, 2009), and most recently the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR ( Cong et al., 2013; Mali et al., 2013a).

Meganuclease, ZF, and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate its nuclease and DNA-binding domains, ZF and TALE proteins consist of individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively ( Figure 2B ). ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of FokI to direct nucleolytic activity toward specific genomic loci. Each of these platforms, however, has unique limitations.

Meganucleases have not been widely adopted as a genome engineering platform due to lack of clear correspondence between meganuclease protein residues and their target DNA sequence specificity. ZF domains, on the other hand, exhibit context-dependent binding preference dueto crosstalk between adjacent modules when assembled into a larger array ( Maeder et al., 2008). Although multiple strategies have been developed to account for these limitations ( Gonzaelz et al., 2010; Sander et al., 2011), assembly of functional ZFPs with the desired DNA binding specificity remains a major challenge that requires an extensive screening process. Similarly, although TALE DNA-binding monomers are for the most part modular, they can still suffer from context-dependent specificity ( Juillerat et al., 2014), and their repetitive sequences render construction of novel TALE arrays labor intensive and costly.

Given the challenges associated with engineering of modular DNA-binding proteins, new modes of recognition would significantly simplify the development of custom nucleases. The CRISPR nuclease Cas9 is targeted by a short guide RNA that recognizes the target DNA via Watson-Crick base pairing ( Figure 2C ). The guide sequence within these CRISPR RNAs typically corresponds to phage sequences, constituting the natural mechanism for CRISPR antiviral defense, but can be easily replaced by a sequence of interest to retarget the Cas9 nuclease. Multiplexed targeting by Cas9 can now be achieved at unprecedented scale by introducing a battery of short guide RNAs rather than a library of large, bulky proteins. The ease of Cas9 targeting, its high efficiency as a site-specific nuclease, and the possibility for highly multiplexed modifications have opened up a broad range of biological applications across basic research to biotechnology and medicine.

The utility of customizable DNA-binding domains extends far beyond genome editing with site-specific endonucleases. Fusing them to modular, sequence-agnostic functional effector domains allows flexible recruitment of desired perturbations, such as transcriptional activation, to a locus of interest ( Xu and Bestor, 1997; Beerli et al., 2000a; Konermann et al., 2013; Maeder et al., 2013a; Mendenhall et al., 2013). In fact, any modular enzymatic component can, in principle, be substituted, allowing facile additions to the genome engineering toolbox. Integration of genome- and epigenome-modifying enzymes with inducible protein regulation further allows precise temporal control of dynamic processes ( Beerli et al., 2000b; Konermann et al., 2013).

CRISPR-Cas9: From Yogurt to Genome Editing

The recent development of the Cas9 endonuclease for genome editing draws upon more than a decade of basic research into understanding the biological function of the mysterious repetitive elements now known as CRISPR ( Figure 3 ), which are found throughout the bacterial and archaeal diversity. CRISPR loci typically consist of a clustered set of CRISPR-associated (Cas) genes and the signature CRISPR array-a series of repeat sequences (direct repeats) interspaced by variable sequences (spacers) corresponding to sequences within foreign genetic elements (protospacers) ( Figure 4 ). Whereas Cas genes are translated into proteins, most CRISPR arrays are first transcribed as a single RNA before subsequent processing into shorter CRISPR RNAs (crRNAs), which direct the nucleolytic activity of certain Cas enzymes to degrade target nucleic acids.

The CRISPR story began in 1987. While studying the iap enzyme involved in isozyme conversion of alkaline phosphatase in E. coli, Nakata and colleagues reported a curious set of 29 nt repeats downstream of the iap gene ( Ishino et al., 1987). Unlike most repetitive elements, which typically take the form of tandem repeats like TALE repeat monomers, these 29 nt repeats were interspaced by five intervening 32 nt nonrepetitive sequences. Over the next 10 years, as more microbial genomes were sequenced, additional repeat elements were reported from genomes of different bacterial and archaeal strains. Mojica and colleagues eventually classified interspaced repeat sequences as a unique family of clustered repeat elements present in >40% of sequenced bacteria and 90% of archaea ( Mojica et al., 2000).

These early findings began to stimulate interest in such microbial repeat elements. By 2002, Jansen and Mojica coined the acronym CRISPR to unify the description of microbial genomic loci consisting of an interspaced repeat array ( Jansen et al., 2002; Barrangou and van der Oost, 2013). At the same time, several clusters of signature CRISPR-associated ( cas) genes were identified to be well conserved and typically adjacent to the repeat elements ( Jansen et al., 2002), serving as a basis for the eventual classification of three different types of CRISPR systems (types I-III) ( Haft et al., 2005; Makarova et al., 2011b). Types I and III CRISPR loci contain multiple Cas proteins, now known to form complexes with crRNA (CASCADE complex for type I; Cmr or Csm RAMP complexes for type III) to facilitate the recognition and destruction of target nucleic acids ( Brouns et al., 2008; Hale et al., 2009) ( Figure 4 ). In contrast, the type II system has a significantly reduced number of Cas proteins. However, despite increasingly detailed mapping and annotation of CRISPR loci across many microbial species, their biological significance remained elusive.

A key turning point came in 2005, when systematic analysis of the spacer sequences separating the individual direct repeats suggested their extrachromosomal and phage-associated origins ( Mojica et al., 2005; Pourcel et al., 2005; Bolotin et al., 2005). This insight was tremendously exciting, especially given previous studies showing that CRISPR loci are transcribed ( Tang et al., 2002) and that viruses are unable to infect archaeal cells carrying spacers corresponding to their own genomes ( Mojica et al., 2005). Together, these findings led to the speculation that CRISPR arrays serve as an immune memory and defense mechanism, and individual spacers facilitate defense against bacteriophage infection by exploiting Watson-Crick base-pairing between nucleic acids ( Mojica et al., 2005; Pourcel et al., 2005). Despite these compelling realizations that CRISPR loci might be involved in microbial immunity, the specific mechanism of how the spacers act to mediate viral defense remained a challenging puzzle. Several hypotheses were raised, including thoughts that CRISPR spacers act as small RNA guides to degrade viral transcripts in a RNAi-like mechanism ( Makarova et al., 2006) or that CRISPR spacers direct Cas enzymes to cleave viral DNA at spacer-matching regions ( Bolotin et al., 2005).

Working with the dairy production bacterial strain Streptococcus thermophilus at the food ingredient company Danisco, Horvath and colleagues uncovered the first experimental evidence for the natural role of a type II CRISPR system as an adaptive immunity system, demonstrating a nucleic-acid-based immune system in which CRISPR spacers dictate target specificity while Cas enzymes control spacer acquisition and phage defense ( Barrangou et al., 2007). A rapid series of studies illuminating the mechanisms of CRISPR defense followed shortly and helped to establish the mechanism as well as function of all three types of CRISPR loci inadaptive immunity. By studying the type I CRISPR locus of Escherichia coli, van der Oost and colleagues showed that CRISPR arrays are transcribed and converted into small crRNAs containing individual spacers to guide Cas nuclease activity ( Brouns et al., 2008). In the same year, CRISPR-mediated defense by a type III-A CRISPR system from Staphylococcus epidermidis was demonstrated to block plasmid conjugation, establishing the target of Cas enzyme activity as DNA rather than RNA ( Marraffini and Sontheimer, 2008), although later investigation of a different type III-B system from Pyrococcus furiosus also revealed crRNA-directed RNA cleavage activity ( Hale et al., 2009, 2012).

As the pace of CRISPR research accelerated, researchers quickly unraveled many details of each type of CRISPR system ( Figure 4 ). Building on an earlier speculation that protospacer adjacent motifs (PAMs) may direct the type II Cas9 nuclease to cleave DNA ( Bolotin et al., 2005), Moineau and colleagues highlighted the importance of PAM sequences by demonstrating that PAM mutations in phage genomes circumvented CRISPR interference ( Deveau et al., 2008). Additionally, for types I and II, the lack of PAM within the direct repeat sequence within the CRISPR array prevents self-targeting by the CRISPR system. In type III systems, however, mismatches between the 5′ end of the crRNA and the DNA target are required for plasmid interference ( Marraffini and Sontheimer, 2010).

By 2010, just 3 years after the first experimental evidence for CRISPR in bacterial immunity, the basic function and mechanisms of CRISPR systems were becoming clear. A variety of groups had begun to harness the natural CRISPR system for various biotechnological applications, including the generation of phage-resistant dairy cultures ( Quiberoni et al., 2010) and phylogenetic classification of bacterial strains ( Horvath et al., 2008, 2009). However, genome editing applications had not yet been explored.

Around this time, two studies characterizing the functional mechanisms of the native type II CRISPR system elucidated the basic components that proved vital for engineering a simple RNA-programmable DNA endonuclease for genome editing. First, Moineau and colleagues used genetic studies in Streptococcus thermophilus to reveal that Cas9 (formerly called Cas5, Csn1, or Csx12) is the only enzyme within the cas gene cluster that mediates target DNA cleavage ( Garneau et al., 2010). Next, Charpentier and colleagues revealed a key component in the biogenesis and processing of crRNA in type II CRISPR systems-a noncoding trans-activating crRNA (tracrRNA) that hybridizes with crRNA to facilitate RNA-guided targeting of Cas9 ( Deltcheva et al., 2011). This dual RNA hybrid, together with Cas9 and endogenous RNase III, is required for processing the CRISPR array transcript into mature crRNAs ( Deltcheva et al., 2011). These two studies suggested that there are at least three components (Cas9, the mature crRNA, and tracrRNA) that are essential for reconstituting the type II CRISPR nuclease system. Given the increasing importance of programmable site-specific nucleases based on ZFs and TALEs for enhancing eukaryotic genome editing, it was tantalizing to think that perhaps Cas9 could be developed into an RNA-guided genome editing system. From this point, the race to harness Cas9 for genome editing was on.

In 2011, Siksnys and colleagues first demonstrated that the type II CRISPR system is transferrable, in that transplantation of the type II CRISPR locus from Streptococcus thermophilus into Escherichia coli is able to reconstitute CRISPR interference in a different bacterial strain ( Sapranauskas et al., 2011). By 2012, biochemical characterizations by the groups of Charpentier, Doudna, and Siksnys showed that purified Cas9 from Streptococcus thermophilus or Streptococcus pyogenes can be guided by crRNAs to cleave target DNA in vitro ( Jinek et al., 2012; Gasiunas et al., 2012), in agreement with previous bacterial studies ( Garneau et al., 2010; Deltcheva et al., 2011; Sapranauskas et al., 2011). Furthermore, a single guide RNA (sgRNA) can be constructed by fusing a crRNA containing the targeting guide sequence to a tracrRNA that facilitates DNA cleavage by Cas9 in vitro ( Jinek et al., 2012).

In 2013, a pair of studies simultaneously showed how to successfully engineer type II CRISPR systems from Streptococcus thermophilus ( Cong et al., 2013) and Streptococcus pyogenes ( Cong et al., 2013; Mali et al., 2013a) to accomplish genome editing in mammalian cells. Heterologous expression of mature crRNA-tracrRNA hybrids ( Cong et al., 2013) as well as sgRNAs ( Cong et al., 2013; Mali et al., 2013a) directs Cas9 cleavage within the mammalian cellular genome to stimulate NHEJ or HDR-mediated genome editing. Multiple guide RNAs can also be used to target several genes at once. Since these initial studies, Cas9 has been used by thousands of laboratories for genome editing applications in a variety of experimental model systems ( Sander and Joung, 2014). The rapid adoption of the Cas9 technology was also greatly accelerated through a combination of open-source distributors such as Addgene, as well as a number of online user forums such as http://www.genome-engineering.org and http://www.egenome.org.

Structural Organization and Domain Architecture of Cas9

The family of Cas9 proteins is characterized by two signature nuclease domains, RuvC and HNH, each named based on homology to known nuclease domain structures ( Figure 2C ). Though HNH is a single nuclease domain, the full RuvC domain is divided into three subdomains across the linear protein sequence, with RuvC I near the N-terminal region of Cas9 and RuvC II/III flanking the HNH domain near the middle of the protein. Recently, a pair of structural studies shed light on the structural mechanism of RNA-guided DNA cleavage by Cas9.

First, single-particle EM reconstructions of the Streptococcus pyogenes Cas9 (SpCas9) revealed a large structural rearrangement between apo-Cas9 unbound to nucleic acid and Cas9 in complex with crRNA and tracrRNA, forming a central channel to accommodate the RNA-DNA heteroduplex ( Jinek et al., 2014). Second, a high-resolution structure of SpCas9 in complex with sgRNA and the complementary strand of target DNA further revealed the domain organization to comprise of an α-helical recognition (REC) lobe and a nuclease (NUC) lobe consisting of the HNH domain, assembled RuvC subdomains, and a PAM-interacting (PI) C-terminal region ( Nishimasu et al., 2014) ( Figure 5A and Movie S1).

Together, these two studies support the model that SpCas9 unbound to target DNA or guide RNA exhibits an autoinhibited conformation in which the HNH domain active site is blocked by the RuvC domain and is positioned away from the REC lobe ( Jinek et al., 2014). Binding of the RNA-DNA heteroduplex would additionally be sterically inhibited by the orientation of the C-terminal domain. Asaresult, apo-Cas9 likely cannot bind nor cleave target DNA. Like many ribonucleoprotein complexes, the guide RNA serves as a scaffold around which Cas9 can fold and organize its various domains ( Nishimasu et al., 2014).

The crystal structure of SpCas9 incomplex with an sgRNA and target DNA also revealed how the REC lobe facilitates target binding. An arginine-rich bridge helix (BH) within the REC lobe is responsible for contacting the 3′ 8-12 nt of the RNA-DNA heteroduplex ( Nishimasu et al., 2014), which correspond with the seed sequence identified through guide sequence mutation experiments ( Jinek et al., 2012; Cong et al., 2013; Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013; Mali et al., 2013b).

The SpCas9 structure also provides a useful scaffold for engineering or refactoring of Cas9 and sgRNA. Because the REC2 domain of SpCas9 is poorly conserved in shorter orthologs, domain recombination or truncation is a promising approach for minimizing Cas9 size. SpCas9 mutants lacking REC2 retain roughly 50% of wild-type cleavage activity, which could be partly attributed to their weaker expression levels ( Nishimasu et al., 2014). Introducing combinations of orthologous domain recombination, truncation, and peptide linkers could facilitate the generation of a suite of Cas9 mutant variants optimized for different parameters such as DNA binding, DNA cleavage, or overall protein size.

Metagenomic, Structural, and Functional Diversity of Cas9

Cas9 is exclusively associated with the type II CRISPR locus and serves as the signature type II gene. Based on the diversity of associated Cas genes, type IICRISPR loci are further subdivided into three subtypes (IIA-IIC) ( Figure 5B ) ( Makarova et al., 2011a; Chylinski et al., 2013). Type II CRISPR loci mostly consist of the cas9, cas1, and cas2 genes, as well as a CRISPR array and tracrRNA. Type IIC CRISPR systems contain only this minimal set of cas genes, whereas types IIA and IIB have an additional signature csn2 or cas4 gene, respectively ( Chylinski et al., 2013).

Subtype classification of type II CRISPR loci is based on the architecture and organization of each CRISPR locus. For example, type IIA and IIB loci usually consist of four cas genes, whereas type IIC loci only contain three cas genes. However, this classification does not reflect the structural diversity of Cas9 proteins, which exhibit sequence homology and length variability irrespective of the subtype classification of their parental CRISPR locus. Of >1,000 Cas9 nucleases identified from sequence databases (UniProt) based on homology, protein length israther heterogeneous, roughly ranging from 900 to 1600 amino acids ( Figure 5C ). The length distribution of most Cas9 proteins can be divided into two populations centered around 1,100 and 1,350 amino acids in length. It is worth noting that a third population of large Cas9 proteins belonging to subtype IIA, formerly called Csx12, typically contain around 1500 amino acids.

Despite the apparent diversity of protein length, all Cas9 proteins share similar domain architecture ( Makarova et al., 2011a; Chylinski et al., 2013, 2014; Fonfara et al., 2014), consisting of the RuvC and HNH nuclease domains and the REC domain, an α-helix-rich region with an Arg-rich bridge helix. Unlike type I and III CRISPR systems, which are found in both bacteria and archaea, type II CRISPRs have so far only been found in bacterial strains ( Chylinski et al., 2013). The majority of Cas9 orthologs in fact belong to the phyla of Bacteroidetes, Proteobacteria, and Firmicutes ( Figure 5D ).

The length difference among Cas9 proteins largely results from variable conservation of the REC domain ( Figure 5E ), which associates with the sgRNA and target DNA. For example, the type IIC Actinomyces naeslundii Cas9, which is more compact than its Streptococcus pyogenes ortholog, has a much smaller REC lobe with substantially different orientation ( Jinek et al., 2014).

Protospacer Adjacent Motif: Cas9 Target Range and Search Mechanism

A critical feature of the Cas9 system is the protospacer-adjacent motif (PAM), which flanks the 3′ end of the DNA target site ( Figure 2C ) and dictates the DNA target search mechanism of Cas9. In addition to facilitating self versus non-self discrimination by Cas9 ( Shah et al., 2013), because direct repeats do not contain PAM sites, biochemical and structural characterization of SpCas9 suggested that PAM recognition is involved in triggering the transition between Cas9 target binding and cleavage conformations ( Sternberg et al., 2014; Jinek et al., 2014; Nishimasu et al., 2014).

Single-molecule imaging indicated that Cas9-crRNA-tracrRNA complexes first associate with PAM sequences throughout the genome, allowing Cas9 to initiate DNA strand separation via unknown mechanisms ( Sternberg et al., 2014). DNA competitor cleavage assays additionally suggested that formation of the RNA-DNA heteroduplex is initiated at the PAM site before proceeding PAM distally by interrogating the target site upstream of the PAM for guide sequence complementarity ( Sternberg et al., 2014). Binding of the PAM and a matching target then triggers Cas9 nuclease activity by activating the HNH and RuvC domains, supported by the observation of HNH domain flexibility within the Cas9-sgRNA-DNA ternary complex ( Nishimasu et al., 2014).

The complexity of the PAM sequences also determines the overall DNA targeting space of Cas9. For example, the 5′-NGG of SpCas9 allows it to target, on average, every 8 bp within the human genome ( Cong et al., 2013; Hsu et al., 2013). Additionally, SpCas9 can target sites flanked by 5′-NAG PAMs ( Jiang et al., 2013; Hsu et al., 2013), albeit at a lower efficiency, further expanding its editing versatility. The PAM is specific to each Cas9 ortholog, even within the same species, such as 5′-NNA GAAW for Streptococcus thermophilus CRISPR1 ( Deveau et al., 2008) and 5′-NGGNG for Streptococcus thermophilus CRISPR3 ( Horvath et al., 2008). Another Cas9 from Neisseria meningitidis with a 5′-NNNNGATT PAM requirement ( Zhang et al., 2013) was recently applied in human pluripotent stem cells ( Hou et al., 2013).

Computational ( Chylinski et al., 2013, 2014; Fonfara et al., 2014) or metagenomic analysis of bacteria and archaea containing CRISPR loci could lead to the discovery of Cas9 nucleases with additional PAMs to expand the targeting range of the Cas9 toolkit. Delivery of multiple Cas9 proteins with different PAM requirements facilitates orthogonal genome engineering, in which independent but simultaneous functions are applied at different loci within the same cell or cell population. NmCas9 and SpCas9, for example, can be employed for independent transcriptional repression and nuclease activity ( Esvelt et al., 2013).

PAM specificity can also be modified. For instance, orthologous replacement of the PAM-interacting (PI) domain from the Streptococcus thermophilus CRISPR3 Cas9 with the corresponding domain from Streptococcus pyogenes Cas9 successfully altered PAM recognition from 5′-NGGNG to 5′-NGG ( Nishimasu et al., 2014). PAM engineering strategies could also be exploited to generate short Cas9 orthologs with flexible 5′-NGG or 5′-NG PAM domains.

Genome Editing Using CRISPR-Cas9 in Eukaryotic Cells

To date, the Streptococcus pyogenes Cas9 (SpCas9) has been used broadly to achieve efficient genome editing in a variety of species and cell types, including human cell lines, bacteria, zebrafish, yeast, mouse, fruit fly, roundworm, rat, common crops, pig, and monkey (see Sander and Joung [2014] for a detailed list). SpCas9 is also dramatically expanding the catalog of genetically tractable model organisms, for example, by enabling the introduction of multiplex mutations in cynomolgus monkeys ( Niu et al., 2014).

SpCas9 can be targeted either with a pair of crRNA and tracrRNA ( Cong et al., 2013) or with a chimeric sgRNA ( Cong et al., 2013; Mali et al., 2013a; Cho et al., 2013; Jinek et al., 2013), as the crRNA or sgRNA contains a 20 nt guide sequence that directly matches the target site. The only requirement for the selection of Cas9 target sites is the presence of a protospacer-adjacent motif (PAM) immediately downstream of the target site.

An early discrepancy in the use of SpCas9 editing of the human genome was the drastically higher levels of NHEJ-induced indels given the same target site, when using the engineered dual guide RNA system ( Cong et al., 2013) compared to the engineered sgRNA(+48) scaffold, which only contained up to the 48th base of tracrRNA. Although sgRNA(+48) is sufficient for cleaving DNA in vitro ( Jinek et al., 2012), extension of the 3′ tracrRNA sequence preserved several hairpin structures (sgRNA(+72) and sgRNA(+84)) that were critical for effective sgRNA-mediated genome editing in vivo ( Mali et al., 2013a; Hsu et al., 2013). The additional stem loops enhance the stability of the sgRNA ( Hsu et al., 2013) and are important for proper Cas9-sgRNA-DNA ternary complex formation ( Nishimasu et al., 2014). These analyses of the sgRNA structure and function indicate that careful sgRNA design is critical for optimal Cas9 activity, especially for testing novel Cas9 candidates derived from metagenomic analysis.

One hallmark of the natural CRISPR-Cas9 system is its inherent ability to efficiently cleave multiple distinct target sequences in parallel ( Barrangou et al., 2007; Garneau et al., 2010) by converting a pre-crRNA transcript containing many spacers into individual guide RNAs duplexes (mature crRNA and tracrRNA) through hybridization with tracrRNA ( Deltcheva et al., 2011). Harnessing this unique aspect of CRISPR interference would enable highly scalable multiplex perturbations. Indeed, coexpression of a CRISPR array containing spacers targeting different genes ( Cong et al., 2013) or a battery of several sgRNAs ( Mali et al., 2013a; Wang et al., 2013) together with SpCas9 has led to efficient multiplex editing in mammalian cells. Surprisingly, CRISPR arrays containing direct repeats interspaced by designer spacers were processed into mature guide RNAs without the introduction of bacterial RNase III. Because RNase III is required for crRNA maturation in prokaryotic cells ( Deltcheva et al., 2011), it is likely that endogenous mammalian RNases play compensatory roles ( Cong et al., 2013).

Specificity of Cas9 Nucleases

Because genome editing leads to permanent modifications within the genome, the targeting specificity of Cas9 nucleases is of particular concern, especially for clinical applications and gene therapy. A combination of in vitro and in vivo assays has been typically used to characterize the specificity of ZFNs and TALENs ( Gabriel et al., 2011), but systematic analysis has remained challenging due to difficulties in synthesizing large libraries of proteins with varying sequence specificity. However, Cas9 target recognition is dictated by the Watson-Crick base-pairing interactions of an RNA guide with its DNA target, enabling experimentally tractable and systematic evaluation of the effect of guide RNA-target DNA mismatches on Cas9 activity.

Streptococcus pyogenes Cas9 specificity has been extensively characterized by multiple groups using mismatched guide RNA libraries, in vitro selection, and reporter assays ( Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013b; Pattanayak et al., 2013). In contrast to previous studies that suggested a seed sequence model for Cas9 specificity, wherein the first 8-12 PAM-proximal guide sequence bases determine specificity ( Jinek et al., 2012; Cong et al., 2013), these studies collectively demonstrate that Cas9 tolerates mismatches throughout the guide sequence in a manner that is sensitive to the number, position, and distribution of the mismatches ( Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013b; Pattanayak et al., 2013). Although the PAM-distal bases of the guide sequence are less important for specificity, meaning that mismatches at those positions often do not abolish Cas9 activity, all positions within the guide contribute to overall specificity. Importantly, off-target sites followed by the 5′-NAG PAM can also lead to off-target cleavage, demonstrating the importance of considering both NGG and NAG PAMs in off-target analysis.

Interestingly, Cas9 requires extensive homology between the guide RNA and target DNA in order to cleave but can remain semi-transiently bound with only a short stretch of complementary sequence between the guide RNA and target DNA. These observations suggest that Cas9 has many off-target binding sites but cleaves only a small fraction of them ( Wu et al., 2014). Thus, concerns about off-target activity could vary widely given a desired application that exploits Cas9 for its DNA binding or cleavage capabilities.

Enzymatic concentration is also an important factor in determining Cas9 off-target mutagenesis. This is particularly important because Cas9 can tolerate even five mismatches within the target site ( Fu et al., 2013). Mismatches appear to be better tolerated when Cas9 is present at high concentrations ( Hsu et al., 2013; Pattanayak et al., 2013), leading to higher off-target activity; decreasing Cas9 concentration significantly improves the on- to off-target ratio at the expense of the efficiency of on-target cleavage ( Hsu et al., 2013). The duration of Cas9 expression is likely an additional factor that tunes off-target activity, though its contributions remain to be carefully investigated.

While potential off-target sites have typically been computationally determined by searching for genomic sequences with high sequence similarity to the desired target locus, wholegenome sequencing or other unbiased ways of labeling DNA DSBs genome-wide may illuminate off-target sites that are not predictable by first-order sequence comparison. Unbiased genome-wide characterizations have been previously used to characterize ZFN off-target mutagenesis ( Gabriel et al., 2011) and could easily be adapted for Cas9 nuclease activity. Such data, perhaps in combination with thermodynamic characterization of guide RNA and target DNA hybridization, will likely provide a quantitative framework for assessing and predicting the off-target activity of Cas9. Multiple groups now provide Cas9 target selection tools (e.g., http://tools.genome-engineering. org, http://zifit.partners.org, and http://www.e-crisp.org).

Improving Cas9 Target Recognition Fidelity

Cas9 nucleases cleave DNA through the activity of their RuvC and HNH nuclease domains, each of which nicks a strand of DNA to generate a blunt-ended DSB ( Figure 2C ). SpCas9 can be converted into a DNA "nickase" that creates a single-stranded break (SSB) by catalytically inactivating the RuvC or HNH nuclease domains ( Gasiunas et al., 2012; Jinek et al., 2012; Sapranauskas et al., 2011) via point mutations ( Figure 6A ). Because DNA single-strand breaks are repaired via the high-fidelity base excision repair (BER) pathway ( Dianov and Hübscher, 2013), Cas9 nickases can be exploited for more specific NHEJ as well as HR.

To improve on-target DSB specificity, a double-nicking approach analogous to dimeric ZFNs or TALENs can be used to increase the overall number of bases that are specifically recognized in the target DNA. Using pairs of guide RNAs ( Mali et al., 2013b; Ran et al.,2013)and an SpCas9 HNH⁺/RuvC − nickase mutant (D10A), properly spaced cooperative nicks can mimic DSBs and mediate efficient indel formation ( Figure 6B ). Because off-target nick sites are precisely repaired, this multiplexed nicking strategy can improve specificity by up to 1,500× relative to the wild-type Cas9 ( Ran et al., 2013).

Because Cas9 nuclease or multiplex nicking activity both stimulate NHEJ, a population of cells cotargeted with a homology donor would eventually possess a mix of indel mutants and donor integrants. Single DNA nicks, however, are also able to mediate donor recombination, albeit at a lower level than with DSBs ( Hsu et al., 2013). Cas9 nickases with single sgRNAs can thus be used to mediate HR rather than NHEJ. Furthermore, off-target integration is highly unlikely due to long homology arms flanking the donor cassette.

In addition to the double-nicking strategy, sgRNAs truncated by 2 or 3 nt have been reported to significantly increase targeting specificity of SpCas9, potentially due to greater mismatch sensitivity ( Fu et al., 2014). These truncated sgRNAs can be combined with multiplex nicking to further reduce off-target mutagenesis ( Fu et al., 2014). Future structure-function analyses and Cas9 and protein engineering via rational design or directed evolution may lead to further improvements in Cas9 specificity.

Applications of Cas9 in Research, Medicine, and Biotechnology

Cas9 can be used to facilitate a wide variety of targeted genome engineering applications. The wild-type Cas9 nuclease has enabled efficient and targeted genome modification in many species that have been intractable using traditional genetic manipulation techniques. The ease of retargeting Cas9 by simply designing a short RNA sequence also enables large-scale unbiased genome perturbation experiments to probe gene function or elucidate causal genetic variants. In addition to facilitating co-valent genome modifications, the wild-type Cas9 nuclease can also be converted into a generic RNA-guided homing device (dCas9) by inactivating the catalytic domains. The use of effector fusions can greatly expand the repertoire of genome engineering modalities achievable using Cas9. For example, a variety of proteins or RNAs can be tethered to Cas9 or sgRNA to alter transcription states of specific genomic loci, monitor chromatin states, or even rearrange the three-dimensional organization of the genome.

Rapid Generation of Cellular and Animal Models

Cas9-mediated genome editing has enabled accelerated generation of transgenic models and expands biological research beyond traditional, genetically tractable animal model organisms ( Sander and Joung, 2014). By recapitulating genetic mutations found in patient populations, CRISPR-based editing could be used to rapidly model the causal roles of specific genetic variations instead of relying on disease models that only phenocopy a particular disorder. This could be applied to develop novel transgenic animal models ( Wang et al., 2013; Niu et al., 2014), to engineer isogenic ES and iPS cell disease models with specific mutations introduced or corrected, respectively, or in vivo and ex vivo gene correction ( Schwank et al., 2013; Wu et al., 2013).

For generation of cellular models, Cas9 can be easily introduced into the target cells using transient transfection of plasmids carrying Cas9 and the appropriately designed sgRNA ( Figure 6C ). Additionally, the multiplexing capabilities of Cas9 offer a promising approach for studying common human diseases-such as diabetes, heart disease, schizophrenia, and autism-that are typically polygenic. Large-scale genome-wide association studies (GWAS), for example, have identified haplotypes that show strong association with disease risk. However, it is often difficult to determine which of several genetic variants in tight linkage disequilibrium with the haplotype or which of several genes in the region are responsible for the phenotype. Using Cas9, one could study the effect of each individual variant or test the effect of manipulating each individual gene on an isogenic background by editing stem cells and differentiating them into cell types of interest.

For generation of transgenic animal models, Cas9 protein and transcribed sgRNA can be directly injected into fertilized zygotes to achieve heritable gene modification at one or multiple alleles in models such as rodents and monkeys ( Wang et al., 2013; Li et al., 2013; Yang et al., 2013; Niu et al., 2014) ( Figure 6D ). By bypassing the typical ES cell targeting stage in generating transgenic lines, the generation time for mutant mice and rats can be reduced from more than a year to only several weeks. Such advances will facilitate cost-effective and large-scale in vivo mutagenesis studies in rodent models and can be combined with highly specific editing ( Fu et al., 2014; Ran et al., 2013) to avoid confounding off-target mutagenesis. Successful multiplex targeting in cynomolgus monkey models was also recently reported ( Niu et al., 2014), suggesting the potential for establishing more accurate modeling of complex human diseases such as neuropsychiatric disorders using primate models. Additionally, Cas9 could be harnessed for direct modification of somatic tissue, obviating the need for embryonic manipulation ( Figure 6E ) as well as enabling therapeutic use for gene therapy.

One outstanding challenge with transgenic animal models generated via zygotic injection of CRISPR reagents is genetic mosaicism, partly due to a slow rate of nuclease-induced mutagenesis. Studies to date have typically relied on the injection of Cas9 mRNA into zygotes (fertilized embryos at the single-cell stage). However, because transcription and translation activity is suppressed in the mouse zygote, Cas9 mRNA translation into active enzymatic form is likely delayed until after the first cell division ( Oh et al., 2000). Because NHEJ-mediated repair is thought to introduce indels of random length, this translation delay likely plays a major role in contributing to genetic mosaicism in CRISPR-modified mice. To overcome this limitation, Cas9 protein and sgRNA could be directly injected into single-cell fertilized embryos. The high rate of nonmutagenic repair by the NHEJ process may additionally contribute to undesired mosaicism because introducing indels that mutate the Cas9 recognition site would then have to compete with zygotic division rates. To increase the mutagenic activity of NHEJ, a pair of sgRNAs flanking a small fragment of the target gene may be used to increase the probability of gene disruption.

Functional Genomic Screens

The efficiency of genome editing with Cas9 makes it possible to alter many targets in parallel, thereby enabling unbiased genome-wide functional screens to identify genes that play an important role in a phenotype of interest. Lentiviral delivery of sgRNAs directed against all genes (either together with Cas9 or to cells already expressing Cas9) can be used to perturb thousands of genomic elements in parallel. Recent papers have demonstrated the ability to perform robust negative and positive selection screens in human cells ( Wang et al., 2014; Shalem et al., 2014) by introducing loss-of-function mutations into early, constitutive coding exons of a different gene in each cell ( Figure 6F ). Genome-wide loss-of-function screens have previously used RNAi, but this approach leads to only partial knockdown, has extensive off-target effects, and is limited to transcribed (and usually protein-coding) genes. By contrast, Cas9-mediated pooled sgRNA screens have been shown to provide increased screening sensitivity as well as consistency and can be designed to target nearly any DNA sequence ( Shalem et al., 2014).

Future applications of single sgRNA libraries may also enable the perturbation of noncoding genetic elements, while multiplex sgRNA delivery may be used to dissect the function of large genomic regions through tiled microdeletions. For example, systematic targeting of gene regulatory regions could facilitate the discovery of distant enhancers, general promoter architectures, and any additional regulatory elements that have an effect on protein levels. An additional application could be to dissect large, uncharacterized genomic regions that are implicated in sequencing studies or GWAS.

Tethering dCas9 to different effector domains may also facilitate genomic screens beyond loss-of-function phenotypes. dCas9 fused to epigenetic modifiers, for instance, could be used to study the effects of methylation or certain chromatin states on cellular differentiation or disease pathologies, whereas transcriptional activators allow screening for gain-of-function phenotypes. Using truncated sgRNAs or building redundancy with several sgRNAs targeting each locus would be important design principles for filtering out false positive signals and improving the interpretability of screening data.

Transcriptional Modulation

dCas9 binding alone to DNA elements may repress transcription by sterically hindering RNA polymerase machinery ( Qi et al., 2013), likely by stalling transcriptional elongation. This CRISPR-based interference, or CRISPRi, works efficiently in prokaryotic genomes but is less effective in eukaryotic cells ( Gilbert et al., 2013). The repressive function of CRISPRi can be enhanced by tethering dCas9 to transcriptional repressor domains such as KRAB or SID effectors, which promote epigenetic silencing ( Gilbert et al., 2013; Konermann et al., 2013). However, dCas9-mediated transcriptional repression needs to be further improved-in the current generation of dCas9-based eukaryotic transcription repressors, even the addition of helper functional domains results in only partial transcriptional knockdown ( Gilbert et al., 2013; Konermann et al., 2013).

Cas9 can also be converted into a synthetic transcriptional activator by fusing it to VP16/VP64 or p65 activation domains ( Figure 6G ). In general, targeting Cas9 activators with a single sgRNA to a particular endogenous gene promoter leads to only modest transcriptional upregulation ( Konermann et al., 2013; Maeder et al., 2013b; Perez-Pinera et al., 2013; Mali et al., 2013b). By tiling a promoter with multiple sgRNAs, several groups have reported strong synergistic effects with nonlinear increases in activation ( Perez-Pinera et al., 2013; Maeder et al., 2013b; Mali et al., 2013b). Although the requirement for multiple sgRNAs to achieve efficient transcription activation is potentially advantageous for increased specificity, screening applications employing libraries of sgRNAs will require highly efficient and specific transcriptional control using individual guide RNAs.

Epigenetic Control

Complex genome functions are defined by the highly dynamic landscape of epigenetic states. Epigenetic modifications that tune histones are thus crucial for transcriptional regulation and play important roles in a variety of biological functions. These marks, such as DNA methylation or histone acetylation, are established and maintained in mammalian cells by a variety of enzymes that are recruited to specific genomic loci either directly or indirectly through scaffolding proteins.

Previously, zinc finger proteins and TAL effectors have been used in a small number of proof-of-concept studies to achieve locus-specific targeting of epigenetic modifying enzymes ( Beerli et al., 2000a; Konermann et al., 2013; Maeder et al., 2013a; Mendenhall et al., 2013). Cas9 epigenetic effectors (epiCas9s) that can artificially install or remove specific epigenetic marks at specific loci would serve as a more flexible platform to probe the causal effects of epigenetic modifications in shaping the regulatory networks of the genome ( Figure 6G ). Of course, the potential for off-target activity or crosstalk between effector domains and endogenous epigenetic complexes would need to be carefully characterized. One solution could be to harness prokaryotic epigenetic enzymes to develop orthogonal epigenetic regulatory systems that minimize crosstalk with endogenous proteins.

Live Imaging of the Cellular Genome

The spatial organization of functional and structural elements within the cell contribute to the functional output of genomes, which can be amplified or suppressed dynamically. However, the way that genomes are modified and how their structural organization in vivo modulates functional output remain unclear. Genomic loci located megabases apart or on entirely different chromosomes could be brought into close proximity given appropriate chromosomal organization, thus mediating long-range trans interactions.

Studying the interactions of specific genes given changing chromatin states would require a robust method to visualize DNA in living cells. Traditional techniques for labeling DNA, such as fluorescence in situ hybridization (FISH), require sample fixation and are thus unable to capture live processes. Fluorescently tagged Cas9 labeling of specific DNA loci was recently developed as a powerful live-cell-imaging alternative to DNA-FISH ( Chen et al., 2013) ( Figure 6H ). Advances in orthogonal Cas9 proteins or modified sgRNAs will build out multi-color and multi-locus capabilities to enhance the utility of CRISPR-based imaging for studying complex chromosomal architecture and nuclear organization.

Inducible Regulation of Cas9 Activity

By exploiting the bilobed structure of Cas9, it may be possible to split the protein into two units and control their reassembly via small-molecule or light-inducible heterodimeric domains ( Figure 6I ). Small-molecule induction would facilitate systemic control of Cas9 in patients or animal models, whereas optical regulation enables more spatially precise perturbation. For example, the light-inducible dimerization domains CIB1 and CRY2 or chemically inducible analogs ABI and PYL, which have been successfully used to construct inducible TALEs (Konermann et al., 2013), may be adapted to engineer inducible Cas9 systems.

Future Development of Cas9-Based Genome Engineering Technologies

Unbiased Analysis of Cas9 Binding and Cleavage

Despite the rapid adoption of Cas9 as a platform technology for genetic and epigenetic perturbation and significant progress in understanding and improving Cas9 specificity, its on- and off-target DNA binding and cleavage profiles still need to be thor oughly evaluated. Studies to date characterizing Cas9 off-target activity have relied on in silico computational prediction or in vitro selection. As a result, they have been unable to account for the likelihood of Cas9 activity that is unpredictable by sequence ho-mology to the sgRNA guide sequence.

Because the off-target activity of dCas9 binding for effector domain localization may be much more extensive than Cas9-mediated genome editing, unbiased profiling methods are needed to refine our understanding of "true positive" Cas9 off-target activity that actually leads to undesired functional outcomes. Cas9-based chromatin immunoprecipitation sequencing (ChIP-seq) analysis at multiple target sites could be a high-throughput solution for understanding binding degen eracy ( Wu et al., 2014), whereas techniques for detecting and labeling double-strand breaks ( Crosetto et al., 2013) will help to achieve a comprehensive map of Cas9-induced off-target indels.

These data together will help to generate predictive models for minimizing off-target activity in gene therapeutics or other applications requiring high levels of precision. Understanding Cas9 binding and cleavage in the context of chromatin accessibility and epigenetic states will also inform better computational evaluation of guide RNA specificity. For example, particular sgRNAs could be evaluated based on the genomic nature of its off-target sites, which would vary by guide sequence. Degenerate targeting of transcriptionally silent genes for a cell type or tissue of interest would likely be preferred to off-target sites in the coding region of essential housekeeping genes.

Although it is still unclear how Cas9 is affected by chromatin accessibility and heterochromatin versus euchromatin, dCas9 transcriptional activators can upregulate transcription at sites lacking DNase I hypersensitivity sites, indicating successful binding to inaccessible chromatin ( Perez-Pinera et al., 2013) ( Figure 6G ). CpG methylation does not appear to affect DNA cleavage in vitro, and Cas9 could introduce indels at a highly methylated promoter in vivo ( Hsu et al., 2013). It will be important to evaluate Cas9 binding and cleavage of genomic loci in relevant primary cells with different chromatin states, ideally in post-mitotic cells in which genomic architecture is stably defined.

Overall, these efforts aimed at improving our understanding of Cas9 binding and cleavage specificity will complement existing methods ( Mali et al., 2013b; Ran et al., 2013; Fu et al., 2014) as well as future protein engineering and metagenomic mining efforts to improve Cas9 specificity and the selection of guide RNA target sites.

Development of Versatile Delivery and Expression Systems for Applications of Cas9

Viral vectors such as adeno-associated virus (AAV) or lentivirus are commonly used for delivering genes of interest in vivo or into cell types resistant to common transfection methods, such as immune cells. AAV vectors have been commonly used for attractive candidates for efficient gene delivery in vivo because of their low immunogenic potential, reduced oncogenic risk from host-genome integration, and well-characterized serotype specificity ( Figure 6E ). However, the most commonly used Cas9 nuclease-encoding gene from Streptococcus pyogenes is >4 kb in length, which is difficult to transduce using AAV due to its 4.7 kb packaging capacity. Non-viral approaches for introducing CRISPR reagents in vivo present a fertile ground for developing novel delivery strategies, from liposomes and aptamers to cell-penetrating peptides and the molecular Trojan horse ( Niewoehner et al., 2014).

However, viral approaches are still highly desirable due to their low immunogenicity and wide array of characterized tropisms. The size constraints of viral vectors can be sidestepped by using significantly smaller Cas9 orthologs derived from metagenomic discovery, several of which have already been characterized and validated in human cells ( Cong et al., 2013; Hou et al., 2013; Esvelt et al., 2013). Interestingly, short Cas9 variants reported to date recognize much longer PAM sequences than SpCas9 (5′-NNAGAAW from Streptococcus thermophilus CRISPR1 or 5′-NNNNGATT from Neisseria meningitidis) ( Zhang et al., 2013; Garneau et al., 2010), whereas some longer orthologs have more relaxed PAMs (5′-NG from Francisella novicida) ( Fonfara et al., 2014). Although the effect of PAM restriction on DNA targeting specificity remains to be investigated, the more limited overall targeting range of short Cas9 variants may be partially compensated for by decreasing the number of potential off-target substrates genome-wide.

Moving beyond Endogenous Cellular Repair

The current generation of genome editing technologies depends on the endogenous DNA repair machinery to introduce loss-of-function mutations or precise modifications ( Figure 2A ). Although Cas9 can be used to generate indel mutations via NHEJ with high efficiency, the absolute rate of HDR remains relatively low. Although it is sufficient for the generation of cell lines, especially when paired with drug selection or FACS enrichment, poor rates of recombination greatly limit the practical utility of Cas9-mediated targeted gene insertion in fertilized zygotes or somatic tissue. Homologous recombination proteins are also mainly expressed in the G2 phase of the cell cycle, making HDR-based gene editing difficult in postmitotic cells such as neurons or cardiac myocytes. As a result, methods for stimulating HDR-based repair or alternative strategies for efficient gene insertion are urgently needed. For instance, the highly efficient DNA damage repair system in Deinococcus radiodurans ( Zahradka et al., 2006) may be exploited to enable efficient genome editing in mitotic as well as postmitotic cells.

Furthermore, the majority of CRISPR-based technology development has focused on the signature Cas9 nuclease from type II CRISPR systems. However, there remains a wide diversity of CRISPR types and functions. Cas RAMP module (Cmr) proteins identified in Pyrococcus furiosus and Sulfolobus solfataricus ( Hale et al., 2012) constitute an RNA-targeting CRISPR immune system, forming a complex guided by small CRISPR RNAs that target and cleave complementary RNA instead of DNA. Cmr protein homologs can be found throughout bacteria and archaea, typically relying on a 5 ′ site tag sequence on the target-matching crRNA for Cmr-directed cleavage.

Unlike RNAi, whichis targeted largely by a 6 nt seed region and to a lesser extent 13 other bases, Cmr crRNAs contain 30-40 nt of target complementarity. Cmr-CRISPR technologies for RNA targeting are thus a promising target for orthogonal engineering and minimal off-target modification. Although the modularity of Cmr systems for RNA-targeting in mammalian cells remains to be investigated, Cmr complexes native to P. furiosus have already been engineered to target novel RNA substrates ( Hale et al., 2009, 2012).

Cas9 as a Therapeutic Molecule for Treating Genetic Disorders

Although Cas9 has already been widely used as a research tool, a particularly exciting future direction is the development of Cas9 as a therapeutic technology for treating genetic disorders. For a monogenic recessive disorder due to loss-of-function mutations (such as cystic fibrosis, sickle-cell anemia, or Duchenne muscular dystrophy), Cas9 may be used to correct the causative mutation. This has many advantages over traditional methods of gene augmentation that deliver functional genetic copies via viral vector-mediated overexpression-particularly that the newly functional gene is expressed in its natural context. For dominant-negative disorders in which the affected gene is haplosufficient (such as transthyretin-related hereditary amyloidosis or dominant forms of retinitis pigmentosum), it may also be possible to use NHEJ to inactivate the mutated allele to achieve therapeutic benefit. For allele-specific targeting, one could design guide RNAs capable of distinguishing between single-nucleotide polymorphism (SNP) variations in the target gene, such as when the SNP falls within the PAM sequence.

Some monogenic diseases also result from duplication of genomic sequences. For these diseases, the multiplexing capability of Cas9 may be exploited for deletion of the duplicated elements. For example, trinucleotide repeat disorders could be treated using two simultaneous DSBs to excise the repeat region. The success of this strategy will likely be higher for diseases such as Friedreich's ataxia, in which duplications occur in noncoding regions of the target gene, because NHEJ-mediated repair may lead to imperfect or frameshifted repair junctions.

In addition to repairing mutations underlying inherited disorders, Cas9-mediated genome editing might be used to introduce protective mutations in somatic tissues to combat nongenetic or complex diseases. For example, NHEJ-mediated inactivation of the CCR5 receptor in lymphocytes ( Lombardo et al., 2007) may be a viable strategy for circumventing HIV infection, whereas deletion of PCSK9 ( Cohenet al., 2005) orangiopoietin ( Musunuru et al., 2010) may provide therapeutic effects against statin-resistant hypercholesterolemia or hyperlipidemia. Although these targets may be also addressed using siRNA-mediated protein knockdown, a unique advantage of NHEJ-mediated gene inactivation is the ability to achieve permanent therapeutic benefit without the need for continuing treatment. As with all gene therapies, it will of course be important to establish that each proposed therapeutic use has a favorable benefit-risk ratio.

Cas9 could be used beyond the direct genome modification of somatic tissue, such as for engineering therapeutic cells. Chimeric antigen receptor (CAR) T cells can be modified ex vivo and reinfused into a patient to specifically target certain cancers ( Couzin-Frankel, 2013). The ease of design and testing of Cas9 may also facilitate the treatment of highly rare genetic variants through personalized medicine. Supporting these tremendous possibilities are a number of animal model studies as well as clinical trials using programmable nucleases that already provide important insights into the future development of Cas9-based therapeutics.

Recently, hydrodynamic delivery of plasmid DNA encoding Cas9 and sgRNA along with a repair template into the liver of an adult mouse model of tyrosinemia was shown to be able to correct the mutant Fah gene and rescue expression of the wild-type Fah protein in ∼1 out of 250 cells ( Yin et al., 2014). In addition, clinical trials successfully used ZF nucleases to combat HIV infection by ex vivo knockout of the CCR5 receptor. In all patients, HIV DNA levels decreased, and in one out of four patients, HIV RNA became undetectable ( Tebas et al., 2014). Both of these results demonstrate the promise of programmable nucleases as a new therapeutic platform.

However, numerous challenges still lie ahead. Most importantly, successful clinical translation will depend on appropriate and efficacious delivery systems to target specific disease tissues. To achieve high levels of therapeutic efficacy and simultaneously address a broad spectrum of genetic disorders, homologous recombination efficiency will need to be significantly improved. Although permanent genome modification has advantages over monoclonal antibody or siRNA treatments, which require repeated administration of the therapeutic molecule, the long-term implications remain unclear. As researchers further develop and test Cas9 toward clinical translation, it will be paramount to thoroughly characterize the safety as well as physiological effects of Cas9 using a variety of preclinical models.

Conclusions

The story of how a mysterious prokaryotic viral defense system became one of the most powerful and versatile platforms for engineering biology highlights the importance of basic science research. Just as recombinant DNA technology benefited from basic investigation of the restriction enzymes that are central to warfare between phage and bacteria, the latest generation of Cas9-based genome engineering tools are also based on components from the microbial antiphage defense system. It is highly likely that the future solutions for efficient and precise gene modification will be found in as of yet unexplored corners of the rich biological diversity of nature.

Acknowledgments

We gratefully acknowledge Sigrid Knemeyer for help with illustration; Ian Slay-maker for structural guidance; Chengwei Luo for expertise in phylogenetic analysis; Emmanuelle Charpentier, Philippe Horvath, Charles Jennings, Ellen Law, Luciano Marraffini, Francisco Mojica, Hiroshi Nishimasu, Virginijus Siksnys, and Alexandro Trevino for discussion and comments; and the CRISPR community for this beautiful story. P.D.H. is a James Mills Pierce Fellow. This work is supported by the NIMH through a NIH Director's Pioneer Award (DP1-MH100706), the NINDS through a NIH Transformative R01 grant (R01-NS 07312401), NSF, the Keck, McKnight, Damon Runyon, Searle Scholars, Klingenstein, Vallee, Merkin, and Simons Foundations, and Bob Metcalfe. CRISPR reagents are available to the academic community through Addgene, and associated protocols, support forum, and computational tools are available via the Zhang lab website ( http://www.genome-engineering.org).

References

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# data[].attributes.attachment_link.title
Development and Applications of CRISPR-Cas9 for Genome Engineering

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Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can ...

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Thyagarajan, B., Olivares, E. C., Hollis, R. P., Ginsburg, D. S., & Calos, M. P. (2001). Site-specific genomic integration in mammalian cells mediated by phage φC31 integrase. Molecular and Cellular Biology, 21(12), 3926-3934.

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Abstract

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

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Feng Y Q, Seibler J, Alami R, Eisen A, Westerman K A, Leblouch P, Fiering S, Bouhassira E. Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange. J Mol Biol. 1999;292:779-785. - PubMed

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national center for biotechnology information

# data[].attributes.attachment_link.title
Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase - PubMed

# data[].attributes.attachment_link.description
We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mous ...

# data[].attributes.body
A edição de genes em embriões humanos tem implicações significativas, pois as alterações são hereditárias.

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Alterações genéticas em organismos podem ter impactos ecológicos não intencionais quando esses organismos são introduzidos na natureza.

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Lander, E. S. (2015). Brave new genome. New England Journal of Medicine, 373(1), 5-8.

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Direct and indirect impacts of synthetic biology on biodiversity conservation.

Macfarlane NBW, Adams J, Bennett EL, Brooks TM, Delborne JA, Eggermont H, Endy D, Esvelt KM, Kolodziejczyk B, Kuiken T, Oliva MJ, Peña Moreno S, Slobodian L, Smith RB, Thizy D, Tompkins DM, Wei W, Redford KH. Macfarlane NBW, et al. iScience. 2022 Oct 20;25(11):105423. doi: 10.1016/j.isci.2022.105423. eCollection 2022 Nov 18. iScience. 2022. PMID: 36388962 Free PMC article. Review.

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Webber, B. L., Raghu, S., & Edwards, O. R. (2015). Opinion: Is CRISPR-based gene drive a biocontrol silver bullet or global conservation threat?. Proceedings of the National Academy of Sciences, 112(34), 10565-10567.

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Scientists have recognized the potential for applying gene drive technologies to the control of invasive species for several years ( 1, 2), yet debate about the application of gene drive has been primarily restricted to mosquitoes ( 3). Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology have restarted discussions of using gene drive for invasive species control ( 4).

The implications are potentially remarkable: for the first time we may genuinely have a tool with the power to permanently eliminate a target species from the planet ( Fig. 1 ). The question is no longer whether we can control invasive species using gene drive, but whether we should. Here we explore the implications of recent developments in CRISPR-Cas9 gene drive technology from a biosecurity perspective, through broad comparison with classical biological control (CBC).

Researchers, policymakers, and resource managers must carefully weigh the risks of implementation that could threaten rather than assist a given ecosystem. To assess the technology's potential and avoid the considerable pitfalls, we must look at the many successes, failures, and cautionary tales born of traditional control methods: they must be precise, must be cognizant of ecosystem-wide implications, and must be wary of and anticipate unintended consequences.

Driven to Extinction

Gene drive technologies provide the ability to disperse engineered genes throughout target populations much more quickly than would be possible via simple genetic inheritance ( 5). In nature, selfish genetic elements use a similar strategy, generating multiple copies across the genome to improve the chances that they are inherited ( 6).

CRISPR-Cas9 is a bacteria-derived endonuclease system that cuts a target DNA sequence based on complementarity to a 20-bp guide RNA (gRNA) ( 7). When included as part of a gene drive, a CRISPR-Cas9-generated mutation will make copies of itself anywhere the genome sequence is complementary to the gRNA ( 7), and hence will convert individuals heterozygous for the mutation into homozygotes ( 4). Unlike previous technologies, CRISPR-Cas9 offers simplicity, flexibility, and precision in gene targeting, such that any standard molecular biology laboratory could adopt the technology with little additional specialist equipment or training ( 7). Perhaps most importantly, CRISPR-Cas9 is efficient and inexpensive compared with all previously proposed gene drive technologies ( 4).

A CRISPR-Cas9 gene drive approach to invasive species control would be based on a laboratory strain with a deleterious trait (e.g., distorted sex ratio, reduced fertility, chemical sensitivity) being mass-reared and released into the field in sufficient numbers for the engineered mutation to spread and control the target population within a desired time frame ( 1). With careful selection of an appropriate coding region, the promulgation of severe deleterious traits could rapidly remove entire populations comprised of individuals with short generation times from both managed and natural environments. In principle, this approach could serve as a "silver bullet" solution for the management of highly threatening invasive alien species. But clearly there are several important factors to take into account, and CBC offers important insights in this context.

Lessons from the Past

To date, CBC has been the only cost-effective management option for controlling widespread and abundant introduced organisms ( 8). Sterile insect technology has also proven to be an effective control option for some target species ( 9), but without gene drive sterile insect technology is not self-propagating and for most targets is cost-prohibitive. We contend that the implementation of a gene drive control strategy against invasive alien species would be highly analogous to a CBC program. The logistics of release will be similar, with stringent prerelease risk assessment of nontarget effects to prevent unintended ecological consequences, followed by large-scale rearing and a distribution strategy based on sound ecological understanding of population dynamics. As such, practitioners of CRISPR-Cas9 gene drive should consider the lessons learned from decades of carefully regulated CBC research if we are to apply this technology to biosecurity challenges. We focus on three relevant priorities: ( i) the importance of understanding target specificity, ( ii) the implications of population connectivity, and ( iii) the need to carefully consider unintended cascades for community dynamics.

First, the strictly regulated process required to approve the introduction of CBC agents requires a systematic testing of agent specificity and the consideration of off-target impacts in the context of phylogenetic relatedness, biogeographic overlap, ecological similarity of a range of nontarget species, and the ecology, evolution, and behavior of the agent ( 8). Any evidence of off-target impacts for CBC represents a risk for agent introduction and is rarely tolerated.

Nontarget effects also need to be an essential consideration for a CRISPR-Cas9 gene drive approach to controlling invasive species ( 4). Off-target mutations in the target genome can be common if the gRNA is poorly designed, because of a tolerance for mismatches (reviewed in ref. 7). Because the CRISPR-Cas9 gene drive technology remains fully functional in the mutated strain after it is created, the chance of off-target mutations also remains and the likelihood increases with every generation pre- and postrelease. If there is any risk of gene flow between the target species and other species, then there is also a risk that the modified sequences could be transferred and the adverse trait manifested in nontarget organisms. If gene drive technology is to be used for biocontrol purposes, we need to have a robust understanding of gene flow networks involving the target species, and an improved understanding of potential specificity limitations for the gRNA.

Population Dispersal

Second, high dispersal ability is a common trait for many invasive alien species, and the risk of long-distance introductions is particularly high for species associated with human movement and trade ( 10, 11). For gene drive, this is seen as advantageous from the point of view of rapid spread of the deleterious mutations throughout the target population. However, many invasive species have been introduced to multiple locations across global trade routes, which effectively act as stepping

Are we are willing to risk the global loss of a species as a result of unintended dispersal of modified individuals back to their native range, to benefit from the control efficiencies that CRISPR-Cas9 gene drive technology could offer?

stones linking alien populations with their native range ( 12). The spread of CBC agents between populations targeted for control or into nontarget populations depends largely on the dispersal traits of the agent, in addition to the risk of anthropogenic or stochastic dispersal ( 13). The critical difference between agent-mediated CBC and a gene drive approach to biocontrol is that the focus for gene drive is the target species itself.

Thus, the spread of a deleterious trait depends on the dispersal ability of the target, as well as anthropogenic or stochastic dispersal. Dispersal of these traits through populations, including into the native range, would be extremely challenging to detect, reducing the ability and increasing the cost of biosecurity measures to prevent spread of modified agents to areas where they remain unwanted. Importantly, targets of CBC are relatively well adapted to the presence of the released agents in their native range (which is generally the agent source), whereas native populations of species targeted by gene drive would remain highly susceptible.

Community Dynamics

Finally, although it might seem that the targeted removal of problematic populations with gene drive technology should only produce positive outcomes, all biosecurity management options have the potential for unintended or indirect ecological consequences that need to be carefully considered. Given the potential for gene drive approaches for eradicating species, the criticism of elevated risks of indirect effects will be more real for such approaches than has been the case with CBC ( 14, 15). There is increasing awareness, however, that with a growing pool of invasive alien species, many will have considerable niche overlap, such that if one introduced species was to be removed from the community, another would be likely to rapidly take its place ( 16, 17). Hence, the overall effect on ecosystem values may remain unchanged even if one invader is eradicated. Furthermore, with long introduction histories also comes change in community structure, with introduced species often fulfilling significant roles, particularly in landscapes with strong anthropogenic modification. As with critical food resources or apex predators filling gaps after earlier human-driven extinctions ( 18), there remains a risk that removing species with gene drive technology could produce unintended cascades that may represent a greater net threat than that of the target species. Given these issues, a consultative and regulated risk-cost-benefit analysis approach, akin to that used in best-practice CBC ( 19), may be a prudent step forward in the use of the gene drive approach in a biosecurity context.

Weighing the Risks

The question is this: Are we are willing to risk the global loss of a species as a result of unintended dispersal of modified individuals back to their native range, to benefit from the control efficiencies that CRISPR-Cas9 gene drive technology could offer? There may be situations where the risks identified above are minimal and the use of this technology for controlling invasive species is deemed acceptable after a full risk-cost-benefit analysis ( Fig. 1 ). Isolated islands facing severe threats from alien reptiles or perhaps rodents (e.g., ref. 20), where border control and physical distance could control any outward gene flow, potentially represent a low risk-high gain priority for action. In contrast, marine invasions, where unintended anthropogenic dispersal remains inappropriately frequent ( 21), or terrestrial invasions of highly effective dispersers such as grasses or volant animals ( 22, 23), could only be viewed as targets with arguably intolerable risk. More challenging situations would include species that are widely viewed as undesirable throughout both their alien and native ranges; yet, in such cases, assumptions about other countries' biosecurity priorities should be handled with great caution. There are many cases where the risks could be deemed insurmountable.

Irrespective of how these biosecurity risks are perceived, we caution that without a regulatory framework that provides a mechanism to work through these issues with clarity and transparency for CRISPR-Cas9 gene drive, this putative silver bullet technology could become a global conservation threat. Biosecurity is just one of many areas in which CRISPR-Cas9 gene drive technology is being focused ( 4). It is encouraging that momentum is building to engage stakeholders and scientists from various disciplines into a public discussion on the potential applications of gene drives (see dels.nas.edu/Study-In-Progress/Gene-Drive-Research-Human/DELS-BLS-15-06). We contend that the extensive experience of regulatory successes (and failures) in the context of CBC can offer an existing framework to provide meaningful guidance for assessing risks and benefits for applications related to invasive species control within this emerging field. The time to develop this regulatory framework is now.

Acknowledgments

We thank Paul Bertsch, Raphael Didham, Gary Fitt, John Scott, and Andy Sheppard for comments on this manuscript.

Footnotes

Any opinions, findings, conclusions, or recommendations expressed in this work are those of the authors and do not necessarily reflect the views of the National Academy of Sciences.

References

3. Alphey L. Genetic control of mosquitoes. Annu Rev Entomol. 2014;59(1):205-224. [[ PubMed] Google Scholar]

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15. Louda SM, Pemberton RW, Johnson MT, Follett PA. Nontarget effects--the Achilles' heel of biological control? Retrospective analyses to reduce risk associated with biocontrol introductions. Annu Rev Entomol. 2003;48(1):365-396. [[ PubMed] Google Scholar]

16. Rouget M, Hui C, Renteria J, Richardson DM, Wilson JRU. Plant invasions as a biogeographical assay: Vegetation biomes constrain the distribution of invasive alien species assemblages. S Afr J Bot. 2015 doi: 10.1016/j.sajb.2015.1004.1009. [[ CrossRef] Google Scholar]

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Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

# data[].attributes.attachment_link.title
Opinion: Is CRISPR-based gene drive a biocontrol silver bullet or global conservation threat?

# data[].attributes.attachment_link.description
Scientists have recognized the potential for applying gene drive technologies to the control of invasive species for several years ( 1, 2), yet debate about the application of gene drive has been primarily restricted to mosquitoes ( 3). Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology have restarted discussions of using gene drive for invasive species control ( 4).

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Em Portugal, a legislação referente à edição genética é influenciada tanto por diretrizes internacionais quanto por legislações europeias, considerando que o país é membro da União Europeia.

O enquadramento legal português aborda a edição genética sob várias perspetivas, incluindo a saúde humana, a biotecnologia e a proteção ambiental.

1. Legislação Relacionada à Saúde Humana:

A edição genética em humanos, especialmente a manipulação da linha germinal (que pode afetar as gerações futuras), é um tema delicado e geralmente restrito por considerações éticas e de segurança.

O enfoque está no consentimento informado, na não discriminação genética e na proteção dos dados genéticos pessoais.

2. Regulamentações sobre Organismos Geneticamente Modificados (OGMs):

Portugal segue as diretivas da União Europeia em relação aos OGMs.

Existe uma regulamentação rigorosa para a libertação e comercialização de OGMs, que inclui avaliações de risco ambiental e à saúde.

3. Pesquisa e Desenvolvimento em Biotecnologia:

A investigação científica em edição genética é permitida, mas está sujeita a regulamentos específicos, que garantem a segurança e ética na pesquisa.

Os projetos de pesquisa devem ser aprovados por comités de ética e devem seguir as normas de segurança biológica.

4. Desafios Atuais:

A evolução rápida das técnicas de edição genética, como CRISPR-Cas9, apresenta desafios regulares, onde a legislação pode não estar completamente adaptada às novas tecnologias.

Há debates em curso sobre os limites éticos e as possíveis aplicações da edição genética, especialmente em áreas controversas como a edição da linha germinal e o melhoramento humano.

5. Envolvimento e Diálogo Público:

Portugal tem vindo a mostrar uma tendência para envolver diferentes partes interessadas, incluindo o público geral, na discussão sobre biotecnologia e edição genética, visando uma tomada de decisão informada e democrática sobre estas questões.

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Beneficência: Garantir benefícios terapêuticos, melhoramento de culturas.

Não maleficência: Prevenir danos potenciais, como efeitos genéticos indesejados.

Autonomia: Respeitar as decisões individuais, incluindo o consentimento informado.

Justiça: Acesso equitativo às tecnologias e consideração das implicações sociais