Reaching New Heights With a Nearly-PAMless Cas

Protospacer Adjacent Motifs (PAMs) are short sequences that are approximately 2–6 base pairs long. PAMs are used in bacteria to detect invading viral sequences, PAM is not found in the bacterial genome and thus serves as a distinguishing factor between the viral and bacterial sequences. Therefore, Cas can detect and cleave foreign sequences without damaging bacterial DNA.

PAMs can reduce the gene-editing capabilities of CRISPR-Cas. In CRISPR editing, the Cas protein can only attach to and cut a gene when there is a PAM nearby. This presents with the problem, that only genes containing a PAM nearby can be edited which limits the gene-editing capabilities significantly. For example, SpCas9 requires the NGG PAM sequence nearby in order to edit genes.

Imagine this scenario: You would like to edit a specific gene with SpCas9 that has disease implications but it doesn't have the specific PAM sequence (NGG) nearby. In this case, researchers would resort to using a Cas variant. The different Cas variants recognise different PAM sequences but even then a lot of the genome still cannot be targeted due to the PAM limitation.

An article was published in Science whereby researchers at Massachusetts General Hospital developed novel SpCas9 variants that apparently are not limited by PAMs and thus have endless gene editing capabilities. The Cas variants described in the publication are called SpG and SpRY. The SpG variant has been engineered to recognise "NGH" PAM sequences and the SpRY variant has been developed to recognise "NHN" PAM sequences making it nearly PAM-less. It was found that these novel Cas variants were able to modify disease-related mutations in regions of the genome that were previously unreachable.

This is all well and good but there could be potential off-target effects. Previous Cas variants that have been developed to have a more relaxed PAM had reduced gene-editing specificity. However, these researchers believe that these novel Cas variants have high fidelity and are comparable to SpCas9, but further research needs to be conducted to understand the novel Cas variants.