Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes

Liyang Zhang; H Tomas Rube; Christopher A Vakulskas; Mark A Behlke; Harmen J Bussemaker; Miles A Pufall

doi:10.1093/nar/gkaa231

Back

Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes

Journal article

Open access

Peer reviewed

Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes

Liyang Zhang, H Tomas Rube, Christopher A Vakulskas, Mark A Behlke, Harmen J Bussemaker and Miles A Pufall

Nucleic acids research, Vol.48(9), pp.5037-5053

05/21/2020

DOI: 10.1093/nar/gkaa231

PMCID: PMC7229833

PMID: 32315032

Files and links (1)

url

https://doi.org/10.1093/nar/gkaa231View

Published (Version of record) Open Access

Abstract

Abstract CRISPR RNA-guided endonucleases (RGEs) cut or direct activities to specific genomic loci, yet each has off-target activities that are often unpredictable. We developed a pair of simple in vitro assays to systematically measure the DNA-binding specificity (Spec-seq), catalytic activity specificity (SEAM-seq) and cleavage efficiency of RGEs. By separately quantifying binding and cleavage specificity, Spec/SEAM-seq provides detailed mechanistic insight into off-target activity. Feature-based models generated from Spec/SEAM-seq data for SpCas9 were consistent with previous reports of its in vitro and in vivo specificity, validating the approach. Spec/SEAM-seq is also useful for profiling less-well characterized RGEs. Application to an engineered SpCas9, HiFi-SpCas9, indicated that its enhanced target discrimination can be attributed to cleavage rather than binding specificity. The ortholog ScCas9, on the other hand, derives specificity from binding to an extended PAM. The decreased off-target activity of AsCas12a (Cpf1) appears to be primarily driven by DNA-binding specificity. Finally, we performed the first characterization of CasX specificity, revealing an all-or-nothing mechanism where mismatches can be bound, but not cleaved. Together, these applications establish Spec/SEAM-seq as an accessible method to rapidly and reliably evaluate the specificity of RGEs, Cas::gRNA pairs, and gain insight into the mechanism and thermodynamics of target discrimination.

Details

Title: Subtitle: Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes
Creators: Liyang Zhang - Integrated DNA Technologies (United States)
H Tomas Rube - University of California, Merced
Christopher A Vakulskas - Integrated DNA Technologies (United States)
Mark A Behlke - Integrated DNA Technologies (United States)
Harmen J Bussemaker - Columbia University
Miles A Pufall - Roy J. and Lucille A. Carver College of Medicine
Resource Type: Journal article
Publication Details: Nucleic acids research, Vol.48(9), pp.5037-5053
DOI: 10.1093/nar/gkaa231
PMID: 32315032
PMCID: PMC7229833
NLM abbreviation: Nucleic Acids Res
ISSN: 0305-1048
eISSN: 1362-4962
Publisher: Oxford University Press
Grant note: DOI: 10.13039/100000002, name: National Institutes of Health, award: R01HG003008, G20RR030893; name: Vagelos Precision Medicine Pilot Program; DOI: 10.13039/100004863, name: NYSTAR, award: C090171; name: NSF-CAREER, award: MCB-1552862
Language: English
Date published: 05/21/2020
Academic Unit: Biochemistry and Molecular Biology
Record Identifier: 9984288716902771

Metrics

6 Record Views

35 Times Cited - Web of Science