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CRISPR Study Details Highly Flexible Cas9 Ortholog, SPAMALOT Pipeline for New Cas9 Discovery

NEW YORK (GenomeWeb) – In a study published online in Science Advances today, researchers from the Massachusetts Institute of Technology described a previously uncharacterized Cas9 ortholog from Streptococcus canis (ScCas9) which has fewer protospacer adjacent motif (PAM) requirements than the more widely used S. pyogenes Cas9 (SpCas9) and is therefore able to target more locations on the genome and more disease-causing mutations.

The researchers also built an automated bioinformatics pipeline, which they called the Search for PAMs by ALignment Of Targets, or SPAMALOT, to further explore the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs.

CRISPR nucleases require PAMs flanking the target sites in order to recognize those sites. Different nucleases have different PAM requirements. SpCas9 requires two G nucleotides as its PAM sequence, which restricts the number of locations it can target to around 9.9 percent of sites on the genome. ScCas9, however, requires only one G nucleotide in a minimal 5'-NNG-3' PAM sequence, the researchers reported.

"CRISPR is like a very accurate and efficient postal system that can reach anywhere you want to go very precisely, but only if the zip code ends in a zero," Joseph Jacobson, the study's senior author and head of the Molecular Machines research group within the MIT Media Lab, said in a statement. "So, it is very accurate and specific, but it limits you greatly in the number of locations you can go to."

The team began by curating all Streptococcus Cas9 protein sequences from the UniProt database, performing global pairwise alignments using the BLOSUM62 scoring matrix and calculating percent sequence homology to SpCas9. ScCas9 had an 89.2 percent sequence homology to SpCas9 as well as a positive-charged insertion of 10 amino acids within the highly conserved REC3 domain. The researchers identified an additional insertion of two amino acids immediately upstream of the two critical arginine residues necessary for PAM binding and hypothesized that these insertions could affect the enzyme's PAM specificity of this enzyme. When they performed experiments to determine the PAM sequences recognized by ScCas9, they found that it could bind to the minimal 5'-NNG-3' PAM, distinct to that of SpCas9's 5'-NGG-3'.

The researchers then went on to compare the PAM specificity of ScCas9 to SpCas9 in human cells by cotransfecting HEK293T cells with plasmids expressing these variants along with single-guide RNAs directed to the VEGFA locus with varying PAM sequences. They found that SpCas9's cleavage activity was impaired at other non–5'-NGG-3' sequences while ScCas9 maintained comparable activity to that of SpCas9 on its 5'-NGG-3' target across all tested targets.

"Overall, these results verify that ScCas9 can serve as an effective alternative to SpCas9 for genome editing in mammalian cells, both at overlapping 5'-NGG-3' and more minimal 5'-NNGN-3' PAM sequences," the authors wrote.

Further, when assessing the accuracy of ScCas9 compared to SpCas9 in three targets, the team found that ScCas9 demonstrated comparable on-target activities for all three, but exhibited negligible off-target activity on the VEGFA site 3 and DNMT1 site 4, and a nearly 1.5-fold decrease in off-to-on target ratio for FANCF site 2, suggesting improved accuracy over SpCas9 on overlapping 5'-NGG-3' targets.

Finally, to establish ScCas9 as a useful genome editing tool, the researchers evaluated its ability to modify a variety of gene targets for several PAM sequences. They constructed sgRNAs to 24 targets within nine endogenous genes in HEK293T cells and evaluated on-target gene modification. "Our results demonstrate that ScCas9 maintains efficiencies comparable to that of SpCas9 on 5'-NGG-3' sequences and on selected 5'-NNG-3' PAM targets, supporting our previous findings," the authors wrote. "Notably, ScCas9 performed less effectively on selected target sequences in the hemoglobin subunit δ (HBD) gene while demonstrating higher efficiencies on 5'-NNG-3' sequences in VEGFA and DNMT1, for example. This variation in efficiency within each PAM group and across different genes indicates that proper target selection within specified genomic regions is critical for successful ScCas9-mediated gene modification."

Success in genome editing with ScCas9 also prompted the team to develop a bioinformatics pipeline for discovering additional Cas9 proteins with novel PAM requirements in the Streptococcus genus. In order to develop SPAMALOT, the investigators mapped a 20-nt portion of spacers flanked by known Streptococcus repeat sequences to candidate protospacers that aligned with no more than two mismatches in phages associated with the genus. They then grouped 12-nt protospacer 3'-adjacent sequences from each alignment by genome and CRISPR repeat and then generated group WebLogos to compute presumed PAM features.

"To date, there are limited open-source tools or platforms specifically for the prediction of PAM sequences, though prior studies have conducted internal bioinformatics-based characterizations before experimental validation," the authors wrote. "Here, we have established SPAMALOT as an accessible resource that we share with the community for application to CRISPR cassettes from other genera. Future development will include broadening the scope of candidate targets beyond genus-associated phage to capture additional sequences that could be beneficial targets, such as lysogens in species that host the same phage."

They also noted that the pipeline could be used to more efficiently to validate and engineer PAM specificities that expand the targeting range of CRISPR, specifically for strictly PAM-constrained technologies such as base editing and homology repair induction.