We are excited to announce Genome Engineering 3.0, a 2-day practical workshop designed to help you use CRISPR-Cas9 system to perform all different types of genome engineering tasks more effectively in your own work. It will be held from May 8-9, 2015 at the Broad Institute (map) in Cambridge, MA, USA. The workshop is open to all and free. For more details, please visit our workshop website, and if you are ready to sign up, you can register here.
CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Three types (I-III) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). To license the associated Cas nuclease for nucleic acid cleavage, the non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). The Type II CRISPR, shown below, is one of the most well characterized systems (see references) and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Wastson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer. For more information on application of CRISPR in mammalian genome engineering, please see our publications (Cong et al. Science 2013), and other references (references).