Engineered CRISPR-Cas9 nucleases with Altered PAM Specificity

Smart overview of the Invention

Engineered CRISPR-Cas9 nucleases have been developed with altered protospacer adjacent motif (PAM) specificities, enhancing their utility in genomic and epigenomic engineering. These modifications allow for more efficient targeting of gene sites, which is crucial for various applications in genetic research and therapy.

Background on CRISPR Technology

CRISPR-Cas9 technology utilizes short RNAs to direct the Cas9 protein to specific DNA sequences, enabling precise genome editing. The requirement for a PAM sequence near the target DNA can limit the range of sites that can be modified. Current widely used Cas9 variants, such as those from Streptococcus pyogenes and Staphylococcus aureus, recognize specific PAM sequences that may not be present in all target genomes.

Engineering Strategies for Enhanced PAM Recognition

Novel engineering approaches were employed to create Cas9 variants that can recognize different PAM sequences. By utilizing structural insights and directed evolution techniques, researchers have developed variants that exhibit improved targeting capabilities in human cells and zebrafish. The engineered variants expand the potential for genome editing by allowing access to previously inaccessible sites.

Characteristics of the Engineered Variants

The engineered SpCas9 proteins feature mutations at key amino acid positions, enhancing their specificity and reducing activity on non-canonical PAMs. Specific mutations, such as those at positions G1104, S1109, and D1135, contribute to the improved performance of these nucleases. Similar modifications have also been made to SaCas9 proteins to broaden their functionality.

Applications and Future Directions

The advancements in engineered CRISPR-Cas9 nucleases hold promise for a wide range of applications, including gene therapy and functional genomics. The ability to target diverse PAM sequences enhances the precision of genome editing techniques, paving the way for more effective treatments for genetic disorders and improved understanding of gene function.