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MGH Researchers Develop Nearly PAMless CRISPR-Cas9 Variant

NEW YORK – Researchers at Massachusetts General Hospital have engineered CRISPR-Cas9 variants that are unconstrained by the requirements of a protospacer adjacent motif (PAM), allowing them to target sites in the genome that were previously inaccessible for editing.

As the researchers wrote in their new study published on Thursday in Science, DNA editing by CRISPR-Cas enzymes requires the recognition of a PAM, which can limit target site recognition to a subset of sequences. To remove this constraint, they used structure-guided engineering to nearly completely relax the NGG PAM requirement of Streptococcus pyogenes Cas9 (SpCas9), developing a variant named SpG capable of targeting an expanded set of NGN PAMs.

They then optimized this enzyme, creating a near-PAMless variant named SpRY, capable of targeting almost all PAMs. It exhibited more robust activities on a wide range of sites with NRN PAMs in human cells, and lower but still 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," the authors wrote. "More broadly, the molecular strategy described herein should in principle be extensible to a wide diversity of Cas orthologs, paving a path toward the development of a suite of editing technologies no longer constrained by their inherent targeting limitations."

The researchers compared the editing activity of SpG to wild-type SpCas9, as well as the previously described xCas9 and SpCas9-NG variants on 78 sites bearing NGNN PAM sequences. They found that the WT SpCas9 had a mean editing activity of 72.8 percent on sites with NGG PAMs and a reduced 4.7 percent mean editing across the remaining NGH sites. Comparatively, SpG exhibited the highest mean editing activities across all NGN PAM sites, averaging 51.2 percent on sites with NGG PAMs and 53.7 percent on sites with NGH PAMs.

The team also investigated whether the improved activities of SpG could enhance the activities of base editors across sites with NGN PAMs. The researchers compared C-to-T editing with WT SpCas9, xCas9, SpCas9-NG, and SpG BE4max cytosine base editor (CBE) constructs across 22 endogenous sites in human cells bearing NGNN PAMs, and observed that whereas WT- and xCas9-CBE exhibited mean C-to-T editing efficiencies above 15 percent only on sites with NGG PAMs, both SpG- and SpCas9-NG-CBE were capable of mean C-to-T editing above 23 percent across NGN sites.

In experiments with adenine base editor (ABE) constructs, the A-to-G editing potencies of WT SpCas9, xCas9, SpCas9-NG, and SpG in the ABEmax architecture across 21 endogenous sites harboring NGNN PAMs were similar as those for CBEs, the researchers said. WT- and xCas9-ABE could only efficiently perform A-to-G edits on target sites with NGG PAMs. However, both SpG- and SpCas9-NG-ABE efficiently edited target sites with NGNN PAMs, and the SpG-ABE exhibited the most robust activity across all NGNN sites.

In experimenting with the SpRY variant, the researchers compared the on-target editing of WT SpCas9 and SpRY across 64 sites, 32 each harboring NANN and NGNN PAMs. They observed that WT SpCas9 preferred NGG>NAG>NGA PAMs with negligible targeting of the remaining NRN PAMs, but that SpRY was more effective than WT at targeting sites encoding NRN PAMs, except for sites harboring canonical NGG PAMs. They also noted that SpRY often exhibited comparable activities to SpG, but that SpG remained the most effective NGN PAM variant.

"In principle, the strategy we utilized to reduce or eliminate the PAM requirement should be applicable to other native or engineered CRISPR-Cas9 and Cas12a orthologs for which structural information is available," the authors concluded. "The potential for undesirable off-target effects requires methods to mitigate them. As observed when developing engineered CRISPR-Cas12a and Cas9 enzymes with expanded PAM tolerances, relaxation of the PAM can reduce specificity. However, both enAsCas12a and SpCas9-NG were compatible with substitutions that enhance genome-wide specificity. Similarly, SpG and SpRY are compatible with SpCas9-HF1 substitutions to eliminate nearly all detectable off-target effects as determined by GUIDE-seq, enabling applications that require higher fidelity." 

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