CRISPR GUARD is a method for reducing off-target editing by Cas9 and base editors. 

Short guide RNAs called "GUARD RNAs" recruit Cas9 complexes to off-target sites but do not permit nuclease activity, thereby protecting them from the mismatched guide RNA by direct competition. CRISPR GUARD Finder searches for guide RNA off-targets and designs GUARD RNAs to protect them from editing.


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:

CRISPR GUARD Finder website


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Sanger Institute Contributors

Photo of Dr Matthew Coelho

Dr Matthew Coelho

Cancer Research UK Career Development Fellow 

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