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CRISPR technologies and the search for the PAM-free nuclease

Collias, Daphne, Beisel, Chase L.·2021·Nature Communications
DOI 10.1038/s41467-020-20633-yPMC PMC7822910
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Abstract

The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.

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  • Cas-12a provides high accuracy at the target gene loci than Cas-9.

    “The Cas-12a protein recognizes a T-rich (5ʹ-TTTN) PAM sequence instead of 5ʹ-NGG and provides high accuracy at the target gene loci than Cas-9.69”

    From Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing (2021) · cites this paper as [69]

  • Mutating either one of the catalytic residues of Cas-9 nuclease (HNH and RuvC) will convert Cas-9 into nickase that could only generate a single-stranded break instead of a blunt cleavage.

    “For instance, mutating either one of the catalytic residues of Cas-9 nuclease (HNH and RuvC) will convert the Cas-9 into nickase that could only generate a single-stranded break instead of a blunt cleavage.69”

    From Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing (2021) · cites this paper as [69]

  • The Cas-12a protein recognizes a T-rich (5ʹ-TTTN) PAM sequence instead of 5ʹ-NGG.

    “The Cas-12a protein recognizes a T-rich (5ʹ-TTTN) PAM sequence instead of 5ʹ-NGG and provides high accuracy at the target gene loci than Cas-9.69”

    From Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing (2021) · cites this paper as [69]

  • Modifying the Cas-9 protein to optimize its nuclease specificity is another way to reduce off-target effects.

    “Modifying the Cas-9 protein to optimize its nuclease specificity is another way to reduce off-target effects.”

    From Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing (2021) · cites this paper as [69]

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Research works printed but not linked: 92

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Metadata

Authors
Collias, D., & Beisel, C. L.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2021-01-22
Volume
12
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-020-20633-y
PMC
PMC7822910
Cited by
304
References
21
Language
en
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