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

Nucleic Acids Research, Jan 2018

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.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://academic.oup.com/nar/article-pdf/46/2/792/23595387/gkx1199.pdf

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

Nucleic Acids Research Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs Daniel E. Ryan 2 David Taussig 2 Israel Steinfeld 2 Smruti M. Phadnis 2 Benjamin D. Lunstad 1 Madhurima Singh 2 Xuan Vuong 2 Kenji D. Okochi 1 Ryan McCaffrey 1 Magdalena Olesiak 0 Subhadeep Roy 1 Chong Wing Yung 2 Bo Curry 2 Jeffrey R. Sampson 2 Laurakay Bruhn 2 Douglas J. Dellinger 1 0 University of Colorado , Boulder, CO , USA 1 Agilent Research Laboratories , Boulder, CO , USA 2 Agilent Research Laboratories , Santa Clara, CA , USA 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 ribosephosphate 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. INTRODUCTION CRISPR–Cas systems evolved to function on relatively small genomes in bacteria, but the recent proliferation of applications in larger genomes has created a growing need for higher specificity ( 1–5 ), especially for applications such as therapeutics where off-target activities have the potential to introduce deleterious effects ( 6,7 ). Various approaches have been reported to increase the specificity of CRISPR– Cas9 to minimize off-target events (8). These include truncations ( 9 ) and extensions ( 10 ) at the 5 ends of gRNAs, colocalization of paired nickase mutants of Cas9 ( 11 ), fusion of catalytically inactive dCas9 to dimerization-dependent Fok1 nuclease ( 12,13 ), and engineered higher-fidelity versions of Cas9 protein ( 14–16 ). Other approaches control the duration of CRISPR activity in eukaryotic cells, for example by transient delivery of Cas9 and gRNA as a ribonucleoprotein complex (gRNP) via cationic lipids or electroporation ( 17,18 ), by inducible temporal control ( 19,20 ), or by timed addition of a CRISPR–Cas9 inhibitor (21) and other strategies. Among these options, transient delivery of gRNP complex has gained widespread favor and the other methods have yet to be broadly adopted. Here, we explore an alternative means of modulating the specificity of CRISPR–Cas systems by incorporating chemical modifications in the 20-nucleotide (nt) guide sequence at the 5 end of a gRNA which mediates the specificity of Cas9 in recognizing and hybridizing complementary protospacers. We have developed robust synthetic chemistry methods to produce single-guide RNAs (sgRNAs) with high fidelity and purity ( 22 ) and have reported that certain chemical modifications in the first and last three terminal nucleotides of sgRNAs can substantially boost CRISPR– Cas9 indel rates and homology-directed repair (HDR) editing events ( 23 ). The terminal modifications are thought to provide resistance to exonucleases, which is especially helpful in primary cells. Other studies exploring the potential advantages of chemical modifications in gRNAs employed crRNA and tracrRNA strands in dual-guide RNA systems and showed that chemical modifications in the crRNA strand can boost indel frequencies in transfected human cells ( 24–26 ), can enable conjugation of donor DNA for template-directed repair ( 27 ), and reportedly can improve the targeting specificity of truncated crRNAs by extensive modification of their ribose-phosphate backbones with 2 -O-methine-4 bridges, phosphorothioates, and 2 -fluoro substituents in specific combinations ( 24 ). In the present work, we chemically synthesized sgRNAs in which we systematically walked individual MP modifications across the 20-nt guide sequence of sgRNAs targeting four different genes, and each sgRNA was complexed with Streptococcus pyogenes Cas9 protein to evaluate the gene editing specificity of the Cas9 sgRNP. Importantly, we identified specific positions in sgRNA guide sequences where MP modification improves cleavage specificity across a variety of target loci and cell types, in several instances improving specificity by an order-of-magnitude or greater while maintaining high on-target activity. MATERIALS AND METHODS sgRNA synthesis RNA oligomers were synthesized on a Dr Oligo 48 synthesizer (Biolytic Lab Performance, Inc. (...truncated)


This is a preview of a remote PDF: https://academic.oup.com/nar/article-pdf/46/2/792/23595387/gkx1199.pdf

Ryan, Daniel E, Taussig, David, Steinfeld, Israel, Phadnis, Smruti M, Lunstad, Benjamin D, Singh, Madhurima, Vuong, Xuan, Okochi, Kenji D, McCaffrey, Ryan, Olesiak, Magdalena, Roy, Subhadeep, Yung, Chong Wing, Curry, Bo, Sampson, Jeffrey R, Bruhn, Laurakay, Dellinger, Douglas J. Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs, Nucleic Acids Research, 2018, pp. 792-803, Volume 46, Issue 2, DOI: 10.1093/nar/gkx1199