Friday, December 23, 2016

CRISPR and other enzymes

The Scientists reports the identification of new enzymes to effect CRISPR targeting. Recall that CRISPR is a targeting RNA sequence and the enzyme, such as Cas9 is used to cut and then allow splicing of segments. CRISPR targets the gene position and the enzyme does the cutting. Cas9 does DSB or double stranded breaks. Other enzymes allow for sticky ends.

As The Scientist states:

Banfield’s team searched the genomes for sequences that were both near cas1, which encodes a conserved CRISPR protein, and close to characteristic sequence repeats. The researchers found sequences for Cas9 in two archaeal genomes extracted from the Richmond Mine in Iron Mountain, California. Previously, archaea were known to use class 1 CRISPR systems, but class 2 had only been identified in bacteria. “We don’t really know how it performs, because that has not been achieved in the laboratory yet,” said Banfield. “Archaea have different biology. The fact that [my collaborators] haven’t yet managed to show its function probably means there are components of the system that we don’t yet know about.” The group also uncovered new types of Cas proteins from groundwater and soil bacteria, dubbed CasX and CasY. “They’re really small, especially CasX,” said Banfield. “That means it’s potentially more useful.” CasX is made up of only 980 amino acids, whereas other Cas enzymes are larger. For instance, the commonly used Cas9 from Staphylococcus pyogenes contains 1,368 amino acids, while a smaller one from S. aureus is made up of 1,053 amino acids (CasY is around 1,200 amino acids). “This is important biotechnologically, because if you look at if from the angle of genome editing, the delivery of small genes into cells is much easier than the delivery of large genes,” ... In partnership with UC Berkeley’s Jennifer Doudna, Banfield’s team demonstrated that CasX and CasY are functional. The researchers introduced CRISPR-CasX and CRISPR-CasY into E. coli, finding that they could block genetic material introduced into the cell.

The article appears in Nature.  The article states:

CRISPR-Cas systems provide microbes with adaptive immunity by employing short sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein bound to RNA recognizes and cleaves targeted sequences. The programmable nature of these minimal systems has enabled their repurposing as a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving untapped the vast majority of enzymes from organisms that have not been cultured. Metagenomics, the sequencing of DNA extracted from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identified novel CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet identified. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in E. coli. Interrogation of environmental microbial communities combined with in vivo experiments allows access to an unprecedented diversity of genomes whose content will expand the repertoire of microbe-based biotechnologies.

The targets and capabilities continue to expand. Stay tuned.