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

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 deve...

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Veröffentlicht in:Science (American Association for the Advancement of Science) 2020-04, Vol.368 (6488), p.290-296
Hauptverfasser: Walton, Russell T., Christie, Kathleen A., Whittaker, Madelynn N., Kleinstiver, Benjamin P.
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Sprache:eng
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Zusammenfassung: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.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.aba8853