Abstract
Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.
Highlights
Precise genome editing using CRISPR-Cas[9] is a promising therapeutic avenue for genetic diseases, off-target editing remains a significant safety concern
Bio-layer interferometry (BLI) revealed comparable off-target association kinetics for catalytically inactive Cas[9] complexed with GUARD RNA or mismatched guide RNA (gRNA), with 29 ± 4% slower binding for the GUARD RNA (Fig. 2a), suggesting that competition at off-target loci is feasible
GUARD RNAs were designed as competitive molecules that are truncated versions of the on-target gRNA but incorporate mismatches found at the off-target site (Fig. 2b)
Summary
Precise genome editing using CRISPR-Cas[9] is a promising therapeutic avenue for genetic diseases, off-target editing remains a significant safety concern. The inactive complexes are generated by Cas[9] binding short gRNAs, or GUARD RNAs, with perfect complementary to the off-target site; gRNAs shorter than 16 nucleotides (nt) in length can direct Cas[9] binding but do not support nuclease activity[18,19,20]. This method can be adapted to use catalytically inactive Cas[9] (dCas9), related RNA-guided nucleases, base editors or other sequence-specific DNA binding proteins to form inert complexes occupying off-target loci to render them inaccessible (Fig. 1)
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