RNA-directed Cleavage and Modification of DNA using CasX (CRISPR-CasX)

Patent Status

Additional Patents Pending

Brief Description

The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets.  Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation.  Current CRISPR Cas technologies are based on systems from cultured bacteria, leaving untapped the vast majority of organisms that have not been isolated.  There is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).

UC Berkeley researchers discovered a new type of Cas protein, CasX, from groundwater samples. CasX is short compared to previously identified CRISPR-Cas endonucleases, and thus use of this protein as an alternative provides the advantage that the nucleotide sequence encoding the protein is relatively short.  CasX utilizes a tracrRNA and a guide RNA to perform double stranded cleavage of DNA. The researchers introduced CRISPR-CasX into E. coli, finding that they could block genetic material introduced into the cell.  Further research results indicated that CRISPR-CasX operates in a manner analogous to CRISPR-Cas9, but utilizing an entirely distinct protein architecture containing different catalytic domains.   CasX is also expected to function under different conditions (e.g., temperature) given the environment of the organisms that CasX was expressed in.  Similar to CRISPR Cas9, CasX enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation. 

Suggested uses

• Diagnostics

Advantages

• Functions under different conditions than currently used CRISPR-Cas proteins (e.g., lower temperatures)
• Nucleotide sequence encoding the CasX protein is short

Publications

CasX enzymes comprise a distinct family of RNA-guided genome editors: Jun-Jie Liu, Natalia Orlova, Benjamin L. Oakes, Enbo Ma, Hannah B. Spinner, Katherine L. M. Baney, Jonathan Chuck, Dan Tan, Gavin J. Knott, Lucas B. Harrington, Basem Al-Shayeb, Alexander Wagner, Julian Brötzmann, Brett T. Staahl, Kian L. Taylor, John Desmarais, Eva Nogales & Jennifer A. Doudna, Nature, volume 566, pages218–223 (2019) 

New CRISPR–Cas systems from uncultivated microbes