Fan_Li_whole_thesis.pdf (6.65 MB)
Development of CRISPR/Cas-mediated gene editing in the retina
thesisposted on 2023-05-28, 00:49 authored by Li, F
Many eye diseases have a distinct genetic etiology and collectively, these account for a large proportion of blindness, worldwide. Despite advances in molecular diagnostics and our understanding of genetic etiology, there are no definitive treatments available for many genetic eye diseases. Emerging technologies ‚ÄövÑvÆ such as gene augmentation therapy or stem cell-based replacement therapy ‚ÄövÑvÆ could help restore vision in a number of patients. Recently developed gene-editing techniques also hold great potential for treating blinding or potentially even life-threatening ocular diseases. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas) is an adaptive, prokaryotic immune defense system that coordinates against viral intrusion; it has since been repurposed in a programmable fashion that allows us to very specifically target a gene locus via a user-specified guide RNA. Due to the ease of designing guide RNAs, CRISPR holds great potential for the study or treatment of inherited diseases. There have been a handful of in vivo CRISPR/Cas studies demonstrating the potential of not only modeling various diseases, but also treating genetic disorders or labeling live cells. Crucial considerations regarding somatic gene editing include adequate delivery to target cells in vivo, elimination of off-target effects and improved gene-editing efficacy via different CRISPR/Cas systems. As a proof-of-concept study, the overall aim of my PhD project was to use fluorescent proteins as a model to explore the efficacy of in vivo CRISPR editing in the retina. Based on our previous study using adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas to modify genes in retinal cells in yellow fluorescent protein (YFP) transgenic mice, my PhD study first focused on developing and validating a self-destructive AAV2-mediated CRISPR/Cas system to improve the biosafety of genome editing in vivo. Next, I performed a direct head-to-head comparison of the in vivo gene-editing efficacy of different CRISPR/Cas systems. My last aim was to use in vitro genome-scale CRISPR screens to identify critical genes underlying the ocular cancer, uveal melanoma. To eliminate the possibility of inadvertent off-target effects of CRISPR/Cas9, we designed a novel self-destructing CRISPR/Cas system that disrupts the Cas enzyme itself with an extra guide targeting SpCas9 along with the original YFP-targeting guide. After validation in a YFP-expressing cell line, I tested it in Thy1-YFP transgenic mouse retina using a dual AAV2 vector delivery system: one vector to deliver the SpCas9 transgene, and the other to deliver their cognate sgRNAs against SpCas9 and the target locus (YFP), as well as an mCherry transgene to validate retinal penetration of the AAV. After 8 weeks, the expression of SpCas9 and the efficacy of YFP gene disruption was quantified. SpCas9 messenger RNA (mRNA) was reduced in retinas treated with dual AAV2-mediated-YFP/SpCas9 targeting CRISPR/Cas compared to those treated with YFP-targeting CRISPR/Cas alone. We also showed that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (~85.5% reduction compared to LacZ/SpCas9-targeting CRISPR/Cas) in the transduced retina of Thy1-YFP transgenic mice. Our data suggest that a self-destructive \kamikaze\"-CRISPR/Cas system can be used as a robust tool for genome editing in the retina without compromising on-target efficiency. To determine the most efficacious CRISPR/Cas endonucleases for retinal editing in vivo I designed and constructed YFP-targeting guide RNAs for different CRISPR/Cas systems followed by in vitro validation and sgRNA selection in YFP-expressing cells. I then performed knockout tests across a range of different conditions using the AAV2-based pseudotype AAV7m8 (single or dual vector delivery system) to deliver CRISPR/Cas constructs into retinal cells in CMV-Cre::Rosa26-YFP transgenic mice. For in vitro validation SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 (single and double vector) and CjCas9 in YFP-expressing cells. AAV7m8-mediated delivery of CRISPR/Cas constructs achieved effective transduction into the outer retinal layer and we found that SpCas9 achieved the highest knockout efficacy among all Cas endonucleases in vivo which was consistent with the in vitro result. Other Cas endonucleases were observed to have low editing efficacy or variation in knockout efficacy. Just as CRISPR/Cas can be applied to target a single gene it is also possible to interrogate every possible gene in the genome. This genome-scale CRISPR/Cas screening approach can be used to identify novel gene targets in different cancers. I sought to then identify genes that are essential for uveal melanoma (UM) which is the most common malignant ocular cancer in adults. I employed a GeCKO (genome-wide CRISPR knockout) screening strategy in the UM cell line OCM-1. By identifying the missing guide-RNA library after 12 passages using next-generation sequencing and bioinformatics analysis tools CRISPRAnalyzeR we found 15 genes with three or more targeted sgRNA deletions during selection that are involved in critical biological pathways. By checking these 15 candidate genes based on open-access data from the Cancer Genome Atlas datasets we found that there was elevated expression of the SLC3A2 gene in UM patients in a pan-cancer view setting and that the expression levels of three genes COQ2 MRPL22 and POLR3K are associated with UM patient survival. Our work provides new insights into the molecular mechanisms of UM and may reveal new therapeutic targets for this deadly disease. In summary we developed a novel self-destructive CRISPR/Cas that can be used as a robust tool for genome editing in the retina of a YFP-expressing transgenic mice model. Moreover we found that SpCas9 achieved the most efficient gene modification among the four CRISPR/Cas endonucleases that we tested in AAV7m8-transduced retinas. With a human UM cell line we identified some novel gene targets using a genome-wide CRISPR/Cas9 library screening approach. These studies add new insight to the in vivo applications of CRISPR/Cas gene editing in the retina which is crucial for optimizing the \"clinic ready\" profile of CRISPR/Cas for impending application in the treatment of blinding eye diseases."
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