Streptococcus Canis Cas9 for Tunable Genome Engineering and Regulation

Technology

This technology uses Cas9 from the bacteria Streptococcus canis (ScCas9) to recognize the PAM sequence NNGT instead of the NGG PAM recognized by the commonly used S. pyogenes Cas9 (SpCas9). This PAM sequence is entirely novel and is not recognized by any previously identified Cas9 variants. The amino acid sequence of ScCas9 is highly homologous to SpCas9, differing significantly only in two regions known to play a role in DNA interaction and PAM spacing. Importantly, ScCas9 can be used to efficiently edit DNA in mammalian cells and can be simply integrated into existing Cas9 platforms since it uses the same sgRNA sequences as SpCas9.

Problem Addressed

The CRISPR-Cas9 system has revolutionized genome editing. However, the range of sequences targetable with Cas9 is currently limited by the need for a defined DNA sequence, called a PAM, immediately following the target region. The strict PAM requirements make very precise amino acid changes difficult and makes editing some DNA stretches nearly impossible. Recent work has expanded the panel of PAM sequences available to researchers, but there remains a need to develop Cas9 variants with expanded PAM specificities.

Advantages

• Cas9 variant with novel NNGT PAM recognition sequence
• Increases the number of DNA sequences that can be targeted with Cas9 gene editing
• Simple integration into established Cas9 platforms using same guide RNA sequences
• Demonstrated efficiency in mammalian cells

Publications

Pranam Chatterjee, Noah Jakimo, and Joseph M. Jacobson. "Divergent PAM Specificity of a Highly-Similar SpCas9 Ortholog." doi: https://doi.org/10.1101/258939.