Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering.

Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR-associated (Cas) proteins of the prokaryotic CRISPR-Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5'-NGG-3' PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5'-NGG-3' PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.

Digging into the lesser-known aspects of CRISPR biology.

A long time has passed since regularly interspaced DNA repeats were discovered in prokaryotes. Today, those enigmatic repetitive elements termed clustered regularly interspaced short palindromic repeats (CRISPR) are acknowledged as an emblematic part of multicomponent CRISPR-Cas (CRISPR associated) systems. These systems are involved in a variety of roles in bacteria and archaea, notably, that of conferring protection against transmissible genetic elements through an adaptive immune-like response. This review summarises the present knowledge on the diversity, molecular mechanisms and biology of CRISPR-Cas. We pay special attention to the most recent findings related to the determinants and consequences of CRISPR-Cas activity. Research on the basic features of these systems illustrates how instrumental the study of prokaryotes is for understanding biology in general, ultimately providing valuable tools for diverse fields and fuelling research beyond the mainstream.