Unconstrained genome targeting with near PAMless engineered CRISPR Cas9 variants
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 Engineered Cas variants nearly eliminate the protospacer adjacent motif requirement for DNA targeting CRISPR enzymes. 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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