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RNA-guided endonucleases of the genome editing CRISPR-Cas system, such as Cas9, are very accurate and efficient at sequence modifications and have many potential applications in the fields of medicine, agriculture, and bioenergy. Despite the usefulness of this gene-editing tool, the targeting range of the Cas9 enzyme remains limited due to the requirement of specific sequences called protospacer adjacent motifs (PAM) flanking the target location for site recognition. However, researchers recently discovered a novel Cas9 enzyme that has minimal PAM requirements, expanding the CRISPR-Cas9 target range and providing access to disease specific mutations that have previously been out of reach.
The most widely used Cas9 enzyme comes from Streptococcus pyogenes (SpCas9) and requires a 5’-NGG-3’ PAM sequence for targeting, thus restricting the number of locations it can target to 9.9% of sites on the genome. In order to diversify the targeting ability of CRISPR-Cas9, scientists have sought to discover or engineer new enzymes with modified and less strict PAM requirements. However, only a handful of these enzymes exist. In a study recently published in Science Advances, researchers from MIT characterized a Cas9 enzyme from Streptococcus canis (ScCas9). This Cas9 is very similar in sequence to the commonly used SpCas9, however, ScCas9 requires only one G nucleotide in a minimal 5’-NNG-3’ PAM sequence, greatly expanding the Cas9 targeting range.
The team from MIT investigated all Streptococcus Cas9 protein sequences for similarity to SpCas9 but with less restrictive PAM requirements. ScCas9 had an 89.2% sequence homology to SpCas9, however, contained two additional amino acids near the PAM binding site which they hypothesized would affect PAM specificity. Experiments investigating PAM recognition revealed ScCas9 could bind to the minimal 5’-NNG-3’ PAM sequence compared to the SpCas9 requiring a 5’-NGG-3’ PAM sequence. More importantly, even though the PAM sequences of the two Cas9 enzymes are different, the ScCas9 had comparable cleavage activity in human cells to that of SpCas9 on 5’-NGG-3’ targets. Therefore, ScCas9 is a successful alternative to SpCas9 for certain overlapping sequences and for targeting regions with single nucleotide PAM sequences.
In order to discover new Cas9 enzymes with novel PAM requirements in the Streptococcus genus, the researchers developed an automated bioinformatics pipeline called Search for PAMS by Alignment of Targets (SPAMALOT), which is an open-source tool that allows researchers to search for additional enzymes to expand CRISPR’s targeting range. Discovery of new, less restrictive Cas9 enzymes can be an important tool for certain applications. Using CRISPR-Cas9 to knock out an entire gene is relatively easy considering there are a number of locations a researcher can choose from to target the gene. However, trying to edit a single base within a gene requires a very precise tool to target editing at that specific base. Identifying novel Cas9 enzymes with less restrictive PAM sequences allows the ability to utilize CRISPR-Cas9 gene editing system to target diseases caused by a single mutation potentially leading to the development of effective treatments.