Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs

Abstract

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence (‘guide sequence’) and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.

Functional Profiling of Single CRISPR/Cas9-Edited Human Long-Term Hematopoietic Stem Cells

“…Our method opens up the possibility of studying gene function relationships not only in LT-HSCs, but also in other stem and progenitor cells to uncover cell type specific phenotypes. We believe that the continuous improvement of CRISPR/Cas9 editing efficiency, for example through chemically modified gRNAs [27][28][29] or different Cas9 variants 30,31 , will further improve this approach. In the future, we envision that this method could potentially be adapted for cellular therapies.…”

Therapeutic gene editing in haematological disorders with CRISPR/Cas9

“…The use of sgRNAs truncated at the 5′ end to shorten the region that binds the target site can significantly improve the specificity of the system without sacrificing on‐target gene editing efficiencies (Fu et al , ). In a recent study, researchers incorporated chemically modified nucleotides into the sgRNA at specific sites in the target‐binding region, thereby modulating the relative affinities to on and off target sites (Ryan et al , ). The Cas9 endonuclease has also been optimized for enhanced specificity of genome editing.…”

Suppression of unwanted CRISPR/Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

“…This multipartite target recognition system is imperfect, and most sgRNAs direct significant cleavage and subsequent unwanted editing at off-target sites whose sequence is similar to the target site [2][3][4][5] . Numerous approaches to reduce off-target editing have been devised, yet are hampered by various limitations [6][7][8][9][10][11][12][13][14][15][16][17] . For example, SpCas9 variants with improved specificity have been engineered [18][19][20] .…”