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Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.

ABSTRACT: Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

SUBMITTER: Walton RT

PROVIDER: S-EPMC7297043 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.

Walton Russell T RT Christie Kathleen A KA Whittaker Madelynn N MN Kleinstiver Benjamin P BP

Science (New York, N.Y.) 20200326 6488

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of <i>Streptococcus pyogenes</i> Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser ...[more]

PMID: 32217751

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