Research Article

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

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Science  17 Apr 2020:
Vol. 368, Issue 6488, pp. 290-296
DOI: 10.1126/science.aba8853

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A PAMless base editor

CRISPR-Cas DNA base editing typically requires a specific motif for targeting known as a protospacer-adjacent motif (PAM). This requirement limits the sequences within a genome that can be targeted. Walton et al. engineered specific variants of the Streptococcus pyogenes Cas9 enzyme named SpG and SpRY that could recognize and edit a wider array of PAMs. Using SpRY, the authors were able to correct previously uneditable mutations associated with human disease. Although off-target effects were observed for these engineered Cas enzymes at levels similar to those of the wild-type enzyme, depending on the context, these engineered enzymes widen the potential applications of precision genome editing.

Science, this issue p. 290

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.

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