Cas9 orthologs (Part 33- CRISPR in gene editing and beyond)
Welcome to the 33rd part of the multi-part series on applications of CRISPR in gene editing and beyond.
While the commonly used wildtype (WT) Streptococcus pyogenes SpCas9 can tolerate multiple mismatches in the target sequence, other naturally occurring Cas9 orthologs from Staphylococcus aureus (SaCas9), Neisseria meningitidis, and Campylobacter jejuni are reported to have higher specificity in genome editing compared to SpCas9. Additionally, these orthologous enzymes show different substrate specificities and recognize different PAM sequences.
Staphylococcus aureus Cas9 (SaCas9): For instance, Cas9 of Staphylococcus aureus, abbreviated as SaCas9, has similar levels of gene targeting compared to SpCas9. Unlike SpCas9, which recognizes PAM sequence NGG, SaCas9 recognizes PAM sequence NNGRRT sequence where R is any purine, may be A or G nucleotide.
The longer PAM sequence NNGRRT of SaCas9 also presents some disadvantages. The range of putative target sites would be reduced compared to the SpCas9, which recognizes a more simple and commonly occurring sequence NGG. To address this limitation, mutagenesis of the SaCas9 has been done to generate SaCas9 variants with different PAM specificities. For instance, mutating the amino acids at positions 782, 968, and 1015 generates the KKHSaCas9 variant, which can recognize the NNNRRT PAM sequence. Additionally, the variant shows similar efficiency and specificity as the wild-type SaCas9.
Neisseria meningitidis Cas9 (NmCas9): Cas9 of Neisseria meningitidis, abbreviated as NmCas9, recognizes PAM sequence NNNNGATT. In addition, the guide RNA associated with NmCas9 recognizes a 24 ntd protospacer, which, together with its more complex PAM, implies a high level of specificity for gene editing purposes.
Campylobacter jejuni Cas9 (CjCas9): Cas9 of Campylobacter jejuni, abbreviated as CjCas9, recognizes NNNVRYAC PAM sequence where V is either an A, a G, or a C nucleotide; and Y is either a T or a C nucleotide.
Thus, the use of Cas9 orthologs with different PAM recognition sequences widens the range of potential sequences that can be targeted for gene editing purposes.
If you liked this article and want to know more about applications of CRISPR in gene editing and beyond, click the below links:
