CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs

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 Gu ide RNA A ssisted R eduction of D amage (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.

The development of genome editing tools as powerful techniques with versatile applications in biotechnology and medicine: CRISPR/Cas9, ZnF and TALE nucleases, RNA interference, and Cre/loxP

“…Efforts were made to develop more specific Cas9•sgRNA systems and systems that activate homology-directed repair instead of non-homologous end joining [93,[98][99][100]. Recent approaches reduce off-target effects by co-delivery of short sgRNAs directed against off-target loci [101]. Other strategies involve using Cas9 orthologues from other organisms, using directed evolution and protein engineering to develop improved Cas9 variants or applying different CRISPR/Cas types [60,[102][103][104][105].…”

Improvements in Gene Editing Technology Boost Its Applications in Livestock

“…For instance, the use of CRISPR/Cas9 RNP instead of a plasmid vector, reduced the risk of off-target mutations as RNP is cleared from the cells within 24 hours after transfection ( DeWitt et al, 2017 ). Furthermore, other methods are in development to minimize the off-target effects such as CRISPR Guide RNA Assisted Reduction of Damage (GUARD) that protects off-target sites by co-delivering short gRNAs directed against off-target loci by competition with the on-target gRNA without affecting on-target editing efficiency ( Coelho et al, 2020 ). Nonetheless, it would be appropriate to investigate off-target mutations in animals, embryos or somatic cells as deeply as possible using methods for identification of off-target sites, such as whole genome sequencing (WGS) and whole-exome sequencing (WXS) ( Ishii, 2017 ).…”

2LTRZFP Interacts Specifically to HIV-1 DNA without Off-Target Effects as Determined by Biolayer Interferometry

“…In 2020, Coelho and colleagues have developed CRISPR GUARD strategy to reduce Cas9 off-target effects without decreasing on-target editing efficiency. The results from BLI demonstrated that the majority of off-targets were protected by CRISPR GUARD molecule [ 32 ]. In our study, the off-target effect of 2LTRZFP-GFP was determined to observe whether 2LTRZFP-GFP can exhibit non-specific binding towards human Chromosome using BLI system.…”