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B - Gene Targeting and Gene Correction -> B2 - Gene Targeting and Gene Correction – In Vitro Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

121: Large-Scale GUIDE-seq-2 Profiling Reveals Effects of Human Genetic Variation on Cellular Off-Target Genome Editing Activity

Type: Oral Abstract Session

Presentation Details
Session Title: Genome Editing Therapies & Safety II
Location: Concourse Hall 152 & 153
Start Time: 5/18/2023 14:45
End Time: 5/18/2023 15:00

CRISPR-Cas genome editors hold enormous potential for the treatment of a wide spectrum of genetic diseases and some therapeutic approaches are in ongoing Phase III clinical trials. As gene therapy products that incorporate human genome editing become approved, thousands of patients may to be treated with these new future therapies. An important unanswered question is how individual genetic variation may impact the safety of these treatments. We and others have described examples of point mutations that affect genome editing activity. Here we describe the first, to our knowledge, large-scale experimental studies of the effects of genetic variation within four human populations (ASW, African Ancestry in Southwest USA; CEU, Utah residents with Northern and Western European ancestry; HCB, Han Chinese in Beijing; and MXL, Mexican Ancestry in Los Angeles) on the genome-wide off-target activity of editors. We reasoned that a high-throughput version of GUIDE-seq, a cell-based method for defining nuclease genome-wide activity, could enable the rapid measurement of change in genome editing activity due to genetic variation within on- and off-target sites. We designed and optimized GUIDE-seq-2, a sensitive, streamlined, automated and high-throughput Tn5-tagmentation enabled version of GUIDE-seq which is comparable to the original method and highly reproducible. To determine the impact of single genetic variation on Cas9 off-target activity, using GUIDE-seq-2 we performed a large-scale evaluation of genome-wide off-target activity for six sgRNAs in lymphoblastoid cell lines (LCLs) from ninety-four individuals across four populations characterized by the 1000 genomes project. We measured CRISPR-Cas9 off-target activity across the six target sites and 94 LCL donors using GUIDE-seq-2 aided by an automated liquid handling system. We detected a total of 963 unique off-target sites across the six target sites and 94 LCL donors that we evaluated. Interestingly, we found that 18.7% (180/963) of the off-targets detected, a relatively high proportion, harbored SNPs within at least one individual’s genome. We then modeled the effects of genetic variation on GUIDE-seq-2 read counts (a direct measurement of cellular Cas9 activity) and found 15% (27/180) of genetic variants within off-target sites had significant effects on Cas9 activity (P<0.05). Variants with strong effects on off-target activity were found at expected protospacer adjacent motif (PAM)-proximal and also less likely PAM-distal regions of the off-target sites. In sum, we found approximately 2.8% (27/963) of all detected off-target sites were significantly affected by non-reference genetic variants, with an average of 32.4% (1.1%-93%) of individuals affected per off-target site. Taken together, our results show that genetic variation can strongly impact cellular Cas9 genome-wide off-target activity and highlights the importance of considering how this may affect the safety and efficacy of broadly applicable genome editing therapies.

Cicera R. Lazzarotto1,2, Yichao Li1, Varun Katta1, Elizabeth Urbina Obregon1, Rachael Wood1, Azusa Matsubara1, Yong Cheng1, Shengdar Q. Tsai1

1St. Jude Children’s Research Hospital, Memphis, TN,2Beam Therapeutics, Cambridge, MA
 Y. Li: None.

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