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Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci.

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Bibliographic Details
Title: Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci.
Authors: Lessard, Samuel1,2, Tardif, Jean-Claude1,2, Lettre, Guillaume1,2, Francioli, Laurent3,4, Alfoldi, Jessica3,4, MacArthur, Daniel G.3,4, Ellinor, Patrick T.4,5, Canver, Matthew C.6,7,8,9, Orkin, Stuart H.6,7,8,9,10
Source: Proceedings of the National Academy of Sciences of the United States of America. 12/26/2017, Vol. 114 Issue 52, p11257-11266. 10p.
Subjects: CRISPRs, Human genetic variation, Locus (Genetics), Genome editing, Single nucleotide polymorphisms
Abstract: The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and offtarget activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both onand off-target specificity. Specifically, we utilize 7,444 whole-genome sequences to examine the effect of variants on the targeting specificity of ~3,000 gRNAs across 30 therapeutically implicated loci. We demonstrate that human genetic variation can alter the off-target landscape genome-wide including creating and destroying protospacer adjacent motifs (PAMs). Furthermore, single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) can result in altered on-target sites and novel potent off-target sites,which can predispose patients to treatment failure and adverse effects, respectively; however, these events are rare. Taken together, these data highlight the importance of considering individual genomes for therapeutic genome-editing applications for the design and evaluation of CRISPR-based therapies to minimize risk of treatment failure and/or adverse outcomes. [ABSTRACT FROM AUTHOR]
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Database: Engineering Source
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Abstract:The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and offtarget activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both onand off-target specificity. Specifically, we utilize 7,444 whole-genome sequences to examine the effect of variants on the targeting specificity of ~3,000 gRNAs across 30 therapeutically implicated loci. We demonstrate that human genetic variation can alter the off-target landscape genome-wide including creating and destroying protospacer adjacent motifs (PAMs). Furthermore, single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) can result in altered on-target sites and novel potent off-target sites,which can predispose patients to treatment failure and adverse effects, respectively; however, these events are rare. Taken together, these data highlight the importance of considering individual genomes for therapeutic genome-editing applications for the design and evaluation of CRISPR-based therapies to minimize risk of treatment failure and/or adverse outcomes. [ABSTRACT FROM AUTHOR]
ISSN:00278424
DOI:10.1073/pnas.1714640114