Engineered CRISPR-Cas9 nucleases with Altered PAM Specificity

Smart overview of the Invention

The patent application discusses engineered CRISPR-Cas9 nucleases with altered Protospacer Adjacent Motif (PAM) specificities, enhancing their utility in genomic and epigenomic engineering. By modifying the PAM recognition capabilities of Cas9 proteins, the invention broadens their applicability for genome targeting across different organisms. This advancement is particularly significant for editing genes in organisms like zebrafish and human cells where traditional Cas9 variants are less effective.

Background

CRISPR-Cas9 nucleases are pivotal tools in genome editing, driven by their ability to be programmed to target specific DNA sequences. The targeting process involves a single guide RNA (sgRNA) that pairs with the DNA target, but efficient cleavage also requires recognition of a PAM sequence adjacent to the target site. The commonly used Streptococcus pyogenes Cas9 (SpCas9) recognizes an NGG PAM, limiting its targeting range. Other Cas9 variants like Staphylococcus aureus Cas9 (SaCas9) recognize longer PAM sequences, which occur less frequently in DNA.

Technical Advances

The invention employs structural insights and directed evolution to engineer SpCas9 and SaCas9 variants with novel PAM specificities. These engineered variants enable precise editing of genetic sites previously inaccessible with wild-type Cas9 proteins. Notably, these modified nucleases demonstrate efficient performance in both human cells and zebrafish models, showcasing their potential for diverse biological applications.

Specific Mutations

The engineered SpCas9 variants include mutations at positions such as G1104, S1109, L1111, D1135, G1218, N1317, R1335, and T1337. Specific alterations like D1135V/R1335Q/T1337R (VQR variant) and D1135E/R1335Q/T1337R (EQR variant) enhance PAM specificity. Similarly, SaCas9 variants feature mutations at positions E735, E782, K929, N968, R1015, A1021, and K1044, with combinations like E782K/N968K/R1015H (KKH variant) improving targeting capabilities.

Applications and Implications

These engineered CRISPR-Cas9 nucleases offer expanded possibilities for genome editing by overcoming limitations posed by PAM constraints. They facilitate precise genetic modifications in research and therapeutic contexts, including allele-specific alterations and targeting small genetic elements such as transcription factor binding sites. The advancements presented in this patent application underscore a significant leap forward in the field of genomic engineering.