bioRxiv Biochemistry
bioRxiv Biochemistry
4 years ago

Chimeric CRISPR guides enhance Cas9 target specificity

P., N., Jacobson, J. M., Chatterjee, Jakimo
Oligonucleotide-guided nucleases (OGNs) have enabled rapid advances in precision genome engineering. Though much effort has gone into characterizing and mitigating mismatch tolerance for the most widely adopted OGN, Streptococcus pyogenes Cas9 (SpCas9), potential off-target interactions may still limit applications where on-target specificity is critical. Here we present a new axis to control mismatch sensitivity along the recognition-conferring spacer sequence of SpCas9's guide RNA (gRNA). We introduce mismatch-evading lowered-thermostability guides (melt-guides) and exhibit how nucleotide-type substitutions in the spacer can reduce cleavage of sequences mismatched by as few as a single base pair. Co-transfecting melt-guides into human cell culture with an exonuclease involved in DNA repair, we observe indel formation on a standard genomic target at approximately 70% the rate of canonical gRNA and undetectable on off-target data.

Publisher URL: http://biorxiv.org/cgi/content/short/147686v1

DOI: 10.1101/147686

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.