Seed sequences mediate off-target activity in the CRISPR-interference system.

Autor:	Rohatgi N; Genentech Computational Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA; Roche Informatics, Hoffman-La Roche Canada, 7070 Mississauga Road, Mississauga, ON, Canada., Fortin JP; Genentech Computational Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA., Lau T; Department of Discovery Oncology, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA., Ying Y; Department of Physiological Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA., Zhang Y; Genentech Computational Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA., Lee BL; Department of Physiological Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA., Costa MR; Department of Discovery Oncology, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: mcosta@gene.com., Reja R; Genentech Computational Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA; Department of Physiological Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: rejar@gene.com.
Jazyk:	angličtina
Zdroj:	Cell genomics [Cell Genom] 2024 Nov 13; Vol. 4 (11), pp. 100693. Date of Electronic Publication: 2024 Nov 06.
DOI:	10.1016/j.xgen.2024.100693
Abstrakt:	The CRISPR interference (CRISPRi) system is a powerful tool for selectively and efficiently silencing genes in functional genomics research applications. However, its off-target activity has not been systematically investigated. Here, we utilized a genome-wide CRISPRi-Cas9 single-guide RNA (sgRNA) library to investigate the presence of off-target activity and its effects on gene expression. Our findings suggest that off-target effects in CRISPRi are quite pervasive and have direct and indirect impacts on gene expression. Most of the identified off-targets can be accounted for by complementarity of the protospacer adjacent motif (PAM)-proximal genomic sequence with the 3' half of the sgRNA spacer sequence, the seed sequence. We also report that while the stability of off-target binding is primarily driven by the PAM-proximal seed sequences, variations in the length of these seed sequences and the degree of mismatch tolerance at various positions can differ across different sgRNAs. Competing Interests: Declaration of interests N.R., J.-P.F., T.L., M.R.C., B.L.L., and R.R. are current employees of Roche/Genentech. Y.Z. is an employee of Scribe Therapeutics, and Y.Y. is an employee of Sana Biotechnology. (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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