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Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility


Schmidheini, Lukas; Mathis, Nicolas; Marquart, Kim Fabiano; Rothgangl, Tanja; Kissling, Lucas; Böck, Desirée; Chanez, Christelle; Wang, Jingrui Priscilla; Jinek, Martin; Schwank, Gerald (2024). Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility. Nature Chemical Biology, 20(3):333-343.

Abstract

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N$_{4}$AH and N$_{5}$HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N$_{3}$VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.

Abstract

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N$_{4}$AH and N$_{5}$HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N$_{3}$VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry

04 Faculty of Medicine > Institute of Pharmacology and Toxicology
07 Faculty of Science > Institute of Pharmacology and Toxicology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Molecular Biology
Life Sciences > Cell Biology
Language:English
Date:1 March 2024
Deposited On:11 Dec 2023 11:51
Last Modified:29 Jun 2024 01:40
Publisher:Nature Publishing Group
ISSN:1552-4450
OA Status:Closed
Publisher DOI:https://doi.org/10.1038/s41589-023-01427-x
PubMed ID:37735239