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Examples of using CRISPR/Cas9 for other mutations or retinal diseases

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Examples of using CRISPR/Cas9 for other mutations or retinal diseases

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Diakatou et al. found that CRSIPR/Cas9 also shows promise to treat NR2E3-associated autosomal dominant RP.²¹

Xu, Park & Tsang' review found that CRISPR/Cas 9 proved effective for the treatment of several retinal diseases such as age-related macular degeneration.²²

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1. Giannelli, S. G. et al. Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery. Human Molecular Genetics 27, 761–779 (2018).
2. Unknown author. Retinitis Pigmentosa. National Eye Institute. https://www.nei.nih.gov/learn-about-eye-health/eye-conditions-and-diseases/retinitis-pigmentosa (2019)
3. Unknown creator. White board: Retinitis Pigmentosa (RP). YourEKA science. https://www.youtube.com/watch v=hHiu3cTMgMs (2016)
4. Woods, K.N., Pfeffer, J. Conformational perturbation, allosteric modulation of cellular signaling pathways, and disease in P23H rhodopsin. Sci Rep 10, 2657 (2020).
5. Lewin, A.S., Drenser, K.A., Hauswirth, W.W., Nishikawa, S., Yasumura, D., Flannery, J.G. & LaVail, M.M. Ribosome rescue of photoreceptor cells in P23H a transgenic rat model of autosomal dominant retinitis pigmentosa. Nat. Med., 4, 967 971 (1998)

6. LaVail, M.M. et al. Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long term survival and late-stage therapy. Proc. Natl. Acad. Sci. U. S. A., 97, 11488-11493 (2000).

7. Mao, H., Gorbatyuk, M.S., Rossmiller, B., Hauswirth, W.W. & Lewis, A.S.  Long-term rescue of retinal structure and function by rhodopsin RNA replacement wih a single adeno-associated viral vector in P23H RHO transgenic mice. Hum. Gene Ther., 23, 356-366 (2012).

8. Tessitore, A. et al. Preferential silencing of a common dominant rhodopsin mutation does not inhibit retinal degeneration in a transgenic model. Mol. Ther., 14, 692-699 (2006).

9. Mussolino, C. et al. Zinc-finger based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa. EMBO. Mol. Med., 3, 118-128 (2011).

10. Applied Biological Materials - abm. CRISPR Cas9 - A Brief Introduction ( Applied Biological Materials - abm, 2014).
11. Applied Biological Materials - abm. 5) CRISPR Cas9 - Screening and Validation Strategies ( Applied Biological Materials - abm, 2018).
12. Molecular cloning of DNA. https://w7.pngwing.com/pngs/86/221/png-transparent molecular-cloning-cell-molecular-biology-angle-text-cell thumbnail.png (PNGWING).

13. Brinkman, E. K., Chen, T., Amendola, M. & Van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research 42, (2014).
14. Young, M. Schematic of the T7EI assay, the basis of the Alt-R Genome Editing Detection Kit. Integrated DNA Technologies (Integrated DNA Technologies, 2017).
15. Bloom, K., Ely, A. & Arbuthnot, P. A T7 Endonuclease I Assay to Detect Talen-Mediated Targeted Mutation of HBV cccDNA. Methods in Molecular Biology Hepatitis B Virus 1540, 85–95 (2016).
16. Matsuda, T. & Cepko, C. L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proceedings of the National Academy of Sciences 101, 16–22 (2004).
17. Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523,481–485 (2015).
18. Mcgrew, D. A. & Hedstrom, L. Towards a Pathological Mechanism for IMPDH1-Linked Retinitis Pigmentosa. Retinal Degenerative Diseases Advances in Experimental Medicine and Biology 539–545 (2012).
19. Sullivan, L. S. et al. Prevalence of Disease-Causing Mutations in Families with Autosomal Dominant Retinitis Pigmentosa: A Screen of Known Genes in 200 Families. Investigative Opthalmology & Visual Science 47, 3052 (2006).
20. Wen, Y. et al. Phenotypic Characterization of 3 Families With Autosomal Dominant Retinitis Pigmentosa Due to Mutations in KLHL7. Archives of Ophthalmology 129, 1475 (2011).
21. Diakatou, M. et al. Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3. International Journal of Molecular Sciences, 22(5), 2607 (2021).
22. Xu, C. L., Park, K. S., & Tsang, S. H. CRISPR/Cas9 genome surgery for retinal diseases. Drug Discovery Today. Technologies, 28, 23–32 (2018).

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An introduction to CRISPE-Cas9 gene editing and NHEJ

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An introduction to CRISPE-Cas9 gene editing and NHEJ

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0:00-2:28 -->Basics of CRISPR-Cas9 gene editing and how it can take advantage of non-homologous end-joining¹⁰

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First time that CRISPR/Cas9 technology was used for allele-specific targeting --> proof-of-concept for application of CRISPR for dominant genetic diseases

Retina is an immune privileged tissue, and therefore well-suited for AAV delivery--> This approach can be applied to other RP-causing mutations as well as other dominant retinal dystrophies such as those caused by mutations in IMPDH1¹⁸, PrPF3¹⁹, KLHL7²⁰ genes.

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Supplemental figure 4B, obtained from Giannelli et al. (1)

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4 potential off-target sites were determined in silico using the web tool Cosmid.
TIDE analysis and clone sequencing after both in vitro and in vivo experiments in mice, didn't detect any cleavage at any of the 4 sites.

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Analysis of main results - Major concerns in RP treatment addressed by Giannelli et al. (1)

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Analysis of main results - Major concerns in RP treatment addressed by Giannelli et al. (1)

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1. To find a treatment strategy for RP that would include specific disruption of the P23H mutant allele while preserving the WT allele;

The designed sgRNA-mMUT -which was only complementary to the P23H allele- ensured specific targeting of the mutant allele without affecting the WT allele

Specific and efficient targeting of the P23H allele in +/P23H genomic background was achieved by Cas9-VQR/sgRNA-mMUT system both in vitro and in vivo.

No off-target cleavage was detected at any of the predicted off-target sites.

2. Said treatment would need to be sufficient to slow photoreceptor degeneration and improve retinal function;

Frameshift mutations leading to premature stop codons in all cleaved sequences disrupted the mutant allele.

Treatment rescued functional activity in photoreceptors.

3. To successfully translate this treatment approach to human cells, by discovering an appropriate mode of delivery that is capable of achieving sufficient transduction in the retina;

NGG PAM (recognized by Cas9-VQR) is conserved in the human Rho gene --> translatability to humans.

IV injection of AAV9-PHP.B encapsulating 2 vectors that will specify Cas9 expression to only rod photoreceptor cells --> method of delivery is translatable to humans.

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Figure 6C-G and Figure 8B-Q obtained from Giannelli et al. (1), edited with Biorender.com

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7. Successful Selective Targeting of Mutant P23H Rho Allele in Mice and in Humans In Vitro 

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7. Successful Selective Targeting of Mutant P23H Rho Allele in Mice and in Humans In Vitro 

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In mice:

1. GFP+ cells through FACs sorting, evaluation of indel frequency in rod photoreceptors show only the P23H Rho allele is affected.

2. FACs sorting of high and low levels of GFP to enrich for editing events show that only the P23H Rho allele is affected and there is a higher accumulation of indels in cells with high GFP levels.

In human cells:

WT and gene-edited RHOP23H/P23H 293T cells are treated with 2 different guides: sgRNA-hWT and -hMUT.

High selectivity and precision for both of the guides in regards to the genotype of the cell.

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Figure 4. Histological and electrophysiological analyses of Rho +/P23H retinae 3 months after the treatment.(1)

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5b. Morphological and functional analyses of retinae 3 months after treatment

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5b. Morphological and functional analyses of retinae 3 months after treatment

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3 months after the treatment, morphological examination of treated and control retinae, as well as transretinal electroretinogram (ERG) recordings were performed. The following results were obtained:

Greater ONL thickness in treated compared to control retinae.

Greater preservation of OS/IS layer length in treated compared to control retinae.

Greater a- and b-wave amplitude in treated compared to control retinae (after normalization, fig U and V) indicating partially rescued phototransduction activity

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Figure 3. Histological analysis of Rho +/P23H retinae 1 month after treatment.(1)

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5a. Morphological analysis of retinae 1 month after treatment

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5a. Morphological analysis of retinae 1 month after treatment

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After histological analysis of non-electroporated (left eye, A) and electroporated (with either Cas9-VQR as control, or Cas9-VQR+sgRNA-mMUT) retinae 1 month after treatment, the following observations were made:

No significant difference in ONL thickness between 3 groups (D).

Significant reduction of OS and IS layer thickness in the control compared to treated retinae.

Misshaping of OS layer in control (I,J) vs. more preservation of OS shape in treated retinae (K,L).

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From 0:00 to 2:36 ³

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Clone sequencing is a highly accurate but slow and labour-intensive method to detect cleavage efficiency following CRISPR gene editing¹. It involves the following steps:

1. PCR amplification of the gene of interest.

2. Molecular cloning of PCR products.

3. Selecting and individually sequencing the clones positive for amplicon of interest. 1

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FYI: a quick video on genomic assays (11)

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FYI: a quick video on genomic assays (11)

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5) CRISPR Cas9 - Screening and Validation Strategies

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Electroporation is a method of gene delivery that uses an electric pulse to physically create a temporary pore in the cell membrane¹⁶. This method is fast and doesn't involve any limitations for the size of vectors. However a major disadvantage is the risk of cell death due to high-voltage electric pulses.¹⁶

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Figure 6A obtained from Giannelli et al. (1), edited with Biorender.com

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2 Vectors: Selective Targeting of Rod Photoreceptors 

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2 Vectors: Selective Targeting of Rod Photoreceptors 

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Figure 5A (top) and figure 5M (bottom) obtained from Giannelli et al. (1)

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6. IV injection of AAV9-PHP.B efficiently transduces through the whole retina

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6. IV injection of AAV9-PHP.B efficiently transduces through the whole retina

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AAV delivery of gene therapy has been proven to be effective in retinal diseases in clinical and preclinical trials. AAV9-PHP.B is an engineered synthetic capsid that:

shows high transduction levels in neural tissues,

overcomes blood-brain-barrier and,

infect a large area in the neuraxis

The capsid was equipped with a CBA-GFP vector cassette and they saw that all retinal cellular subtypes were targeted to different extents, significantly in rod photoreceptors and amacrine cells.

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Figure 1G (top left), figure 1E (top right), and figure 2A (bottom) obtained from Giannelli et al. (1)

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3. Disruption of the mutant allele through frameshift mutations

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3. Disruption of the mutant allele through frameshift mutations

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The most common sequences found at the level of P23H allele in transduced retinal cells, included +1 and +2 frameshift mutations (figure 1G). The most common sequence involved the insertion of an adenine at position 68 (figure 1E). This indel mutant, referred to as IndelA, along with IndelB constitute the most frequent indel mutations. Both IndelA and IndelB contain premature stop codons (figure 2A) which leads to degradation of their RNA transcripts by nonsense-mediated decay.

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TIDE analysis, obtained from TIDE publication (13)

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Tracking Indels by Decomposition (TIDE) assay can simply and quickly identify and quantify indels that result from NHEJ¹³. It involves 3 simple steps:

1. One pair of standard PCR reactions.

2. One pair of standard capillary ("Sanger") sequencing reactions.

3. Analysis of the two using the TIDE web tool which uses an algorithm that reconstructs the spectrum and frequency of each identified indel. ¹³

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Supplementary figure 6 (left) and figure 1 (right) from Giannelli et al. (1)

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2. Selective targeting of the P23H allele in heterozygous cells

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2. Selective targeting of the P23H allele in heterozygous cells

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Selective targeting of the P23H allele in Rho heterozygous mouse embryonic fibroblasts achieved through lentiviral delivery of Cas9-VQR and sgRNA-mMUT (Supplementary figure 6)

e.g: clone sequencing detected 33% indels in the mutant, and no indels in the WT sequences, after targeting by sgRNA-mMUT.

Selective targeting of the P23H allele in Rho heterozygous retinal cells in mice, achieved through delivery of Cas9-VQR and sgRNA-mMUT via electroporation

e.g: clone sequencing detected 93% indels in the mutant and 8% indels in WT sequences.

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Supplementary figure 1 from Giannelli et al. (1)

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1. Targeting the P23H Rho allele using CRISPR

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1. Targeting the P23H Rho allele using CRISPR

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PAM domains in the vicinity of the P23H mutation, and their corresponding sgRNA sequences were identified.
The NGA PAM, which is recognized by Cas9-VQR, and the corresponding sgRNA-mMUT were chosen to be used in the study.
Other PAMs and their corresponding sgRNAs were excluded either due to lack of conservation in the murine Rho gene, or their inability to achieve efficient targeting of the P23H allele in preliminary experiments conducted on mouse embryonic fibroblasts (MEFs).

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1. To find a treatment strategy for RP that would include specific disruption of the P23H mutant allele while preserving the WT allele.

2. Said treatment would need to be sufficient to slow photoreceptor degeneration and improve retinal function.

3. To successfully translate this treatment approach to human cells, by discovering an appropriate mode of delivery that is capable of achieving sufficient transduction in the retina.

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Kleinsteiver et al.¹⁷ induced site-directed mutations in the PAM-interacting domain of Cas9 in order to alter its PAM specifity. One altered Cas9 with 3 site-directed mutations (D1135V , R1335Q, T1337R) demonstrated an enhanced ability to recognize and bind NGA PAM domains (instead of NGG).¹⁷

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T7 endonuclease 1 (T7E1) assay is a fast and inexpensive method to estimate the frequency of indels that result from NHEJ ¹⁵. T7E1 assay includes the following steps:

1. Amplification of the genomic region of interest from targeted cells (may contain indel-containing DNA as well as WT DNA) by PCR

2. DNA is denatured and reannealed again to form either a DNA homoduplex or a heteroduplex at random. DNA heteroduplexes contain a WT strand and an indel-containing strand, therefore carrying a mismatch.

3. DNA duplexes are incubated with the T7 endonuclease enzyme which cleaves only DNA heteroduplexes containing a mismatch.

4. All fragments are resolved on gel by electrophoresis. Cleaved indel-containg sequences result in two or multiple bands, while WT strands result in a single band.

5. Indel frequency can be quantified by measuring band intensity.¹⁵

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What happens because of this gain-of-function mutation?

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What happens because of this gain-of-function mutation?

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Unlike the wild-type rhodopsin, the P23H mutant rhodopsin protein doesn't function as a monomer. Instead the mutation causes a conformational change which results in rhodopsin adopting a homodimerization motif. This can thus be seen as a gain of structural function. The homodimerization disturbs the normal receptor activity of rhodopsin and leads to photo-toxicity.⁴

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We believe that using Crispr/Cas9 for specific inactivation of the P23H mutant allele shows a lot of promise to be used in patients as it slows down photoreceptor degeneration and improves retinal function. However - before taking this approach to the clinic - more research is needed to establish the safety of this approach in humans and gene therapy laws must be amended to include gain-of-function mutations. Then, if these future challenges are met, other mutations that cause RP - or other retinal diseases - can perhaps be treated with CRISPR as well, as this proof-of-concept paper shows.

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Belinda --> methods and results, discussion of main results, padlet organization

Diba --> methods and results, discussion of main results, significance of findings

Merel --> introduction, strengths & weakness, future outlook, examples of using CRISPR/Cas9 for other mutations or retinal diseases, take-home message

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Structure of the retina showing the OS and ONL layers

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How to determine RP disease progression?

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How to determine RP disease progression?

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Outer segment (OS) layer --> shows the first signs of retinal degeneration¹

Outer nuclear layer (ONL) thickness ~ number of photoreceptors¹

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Specific inactivation of the P23H RHO mutant allele through CRISPR/Cas9 can counteract photoreceptor degeneration and RP disease progression

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There is a need for a novel treatment strategy that fights RP disease progression and achieves therapeutic benefits!

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Gene inactivation through:

zinc finger nucleases5, 6

short hairpin RNAs7, 8

ribozymes⁹

Suppression and replacement approach: here both the wildtype and mutant allele were silenced while a functional RHO gene was inserted.¹

These approaches, unfortunately, failed to establish both specificity (little/no off-target effects) and efficacy (high enough RHO levels)...

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An example of a low vision aid (Sight Improvement Center, n.d.)

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Current therapies: ameliorating vision as a symptomatic treatment

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Current therapies: ameliorating vision as a symptomatic treatment

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Unfortunately, the only available options for people with RP are the use of low vision aids or vision rehabilitation. This does not halt nor delay disease progression, however.

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The retina of a healthy person compared to the retina of someone with RP (2)

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primary rod photoreceptor cell death --> night blindness & visual field restriction¹

cone cell deterioration --> loss of central vision¹

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The RHO gene with a P23H point mutation. Figure obtained from Giannelli et al. (1)

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RP is often caused by a mutation in the RHO gene

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RP is often caused by a mutation in the RHO gene

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the most common mutation is the P23H point mutation which converts a C to an A at spot 68 leading to the amino acid histidine. This gain-of-function mutation is autosomal dominant and can be found on the RHO gene ¹

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A hallmark of retinitis pigmentosa is the formation of bone spicules in the eye

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An introduction to... Retinitis Pigmentosa (RP)

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An introduction to... Retinitis Pigmentosa (RP)

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prevalence = 1: 4000

onset in childhood²

most common cause of blindness

diagnosis through:

electroretinogram (ERG)

genetic testing

visual field testing