Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci
Samuel Lessard1,2, Laurent Francioli3,4, Jessica Alfoldi3,4
1Research Center, Montreal Heart Institute, Montréal, QC H1T 1C8, Canada.
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 off-target 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 on- and 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.
Insights
Human genetic variation impacts CRISPR-Cas9 gene editing accuracy. Analyzing whole genomes reveals how variations can affect on- and off-target specificity, influencing therapeutic efficacy and safety.
Area of Science:
- Genetics
- Molecular Biology
- Bioengineering
Background:
- CRISPR-Cas9 gene editing shows promise for treating diseases.
- Understanding on- and off-target activity is crucial for safe and effective CRISPR therapies.
- Current evaluations often overlook the impact of human genetic variation.
Purpose of the Study:
- To investigate how human genetic variation affects CRISPR-Cas9 on- and off-target specificity.
- To assess the influence of variants on guide RNA (gRNA) targeting in therapeutically relevant genes.
- To identify potential risks associated with genetic variations in CRISPR-based therapeutic applications.
Main Methods:
- Analysis of 7,444 whole-genome sequences.
- Examination of approximately 3,000 gRNAs targeting 30 therapeutically implicated loci.
- Assessment of single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) impact on targeting.
Main Results:
- Human genetic variation alters the genome-wide off-target landscape, affecting protospacer adjacent motifs (PAMs).
- SNPs and indels can modify on-target sites and create new off-target sites.
- While rare, these alterations can lead to treatment failure or adverse effects.
Conclusions:
- Individual human genomes must be considered for designing and evaluating CRISPR-based therapies.
- Accounting for genetic variation is essential to minimize risks of treatment failure and adverse outcomes.
- This study underscores the need for personalized approaches in therapeutic genome editing.
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