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

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 cont...

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Bibliographic Details
Published in:Nature chemical biology Vol. 20; no. 3; pp. 333 - 343
Main Authors: Schmidheini, Lukas, Mathis, Nicolas, Marquart, Kim Fabiano, Rothgangl, Tanja, Kissling, Lucas, Böck, Desirée, Chanez, Christelle, Wang, Jingrui Priscilla, Jinek, Martin, Schwank, Gerald
Format: Journal Article
Language:English
Published: New York Nature Publishing Group US 01.03.2024
Nature Publishing Group
Subjects:
631/154/152
631/92/469
Biochemical Engineering
Biochemistry
Biocompatibility
Bioorganic Chemistry
Cell Biology
Chemistry
Chemistry and Materials Science
Chemistry/Food Science
CRISPR
CRISPR-Associated Protein 9 - genetics
CRISPR-Associated Protein 9 - metabolism
CRISPR-Cas Systems - genetics
Directed evolution
Editing
Evolution
Gene Editing
Genetic disorders
Genetic modification
Genome
Genome editing
Genomes
Mutation
Nuclease
Robustness
ISSN:1552-4450, 1552-4469, 1552-4469
Online Access:Get full text
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Summary: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 ( Cj Cas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evo Cj Cas9, 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, evo Cj Cas9 exhibits higher nuclease activity than wild-type Cj Cas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed Sp Cas9 variants. Combined with deaminases or reverse transcriptases, evo Cj Cas9 enables robust base and prime editing, with the small size of evo Cj Cas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems. Through directed evolution, the PAM compatibility of the compact Cas9 variant Cj Cas9 was increased. Evolved Cj Cas9 shows higher nuclease activity at canonical and non-canonical sites and enables robust in vivo gene editing from single AAV vectors.
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ISSN:1552-4450
1552-4469
1552-4469
DOI:10.1038/s41589-023-01427-x