CRISPRs (clustered regularly interspaced short palindromic repeats) and the CRISPR-associated Cas9 nuclease function as part of an adaptive immune system in bacteria and archaea (ISHINO et al. 1987; MAKAROVA et al. 2006; BARRANGOU et al. 2007). In type II CRISPR systems, a CRISPR RNA (crRNA), which contains sequence complementary to invading virus or plasmid DNA, and a trans-activating CRISPR RNA (tracrRNA) interact with Cas9 to direct sequence-specific cleavage of exogenous DNA.
Recognizing the potential of harnessing this system for precise genome engineering in other organisms, Jinek and colleagues identified a minimal two-component system required for the site-specific cleavage of DNA: Cas9 and a chimeric RNA (chiRNA) comprising the crRNA and tracrRNA from S. pyogenes (JINEK et al. 2012). Thus, in this modified CRISPR RNA/Cas9 system a common nuclease is directed to specific DNA sequences by a short, readily generated RNA.
We have found that a variety of Cas9-mediated genome modifications can be efficiently generated in Drosophila and transmitted through the germline. A single chiRNA can guide Cas9 to a specific genomic sequence to induce double strand breaks (DSBs) that are imperfectly repaired by NHEJ. ‘Multiplex’ targeting with two chiRNAs can be used to generate large defined deletions, and Cas9-induced DSBs can mediate gene replacement by homologous recombination.
The ease of producing sequence-specific chiRNAs makes the CRISPR RNA/Cas9 system an appealing method for genome editing. From the initial cloning steps to the identification of transformants, stable lines with targeted genome alterations can be generated within a month – opening the door to rapid engineering of the Drosophila genome to investigate gene function and regulation.
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