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27-09-2017 | Image

Box 1

The CRISPR–Cas system was discovered as a form of immunity in bacteria and adapted for use in genome editing in other organisms ranging from yeast to human cells. As a genome-editing technique, CRISPR is relatively new, but it has rapidly become an important tool for synthetic lethality screening in mammalian cells. The CRISPR–Cas9 ribonucleoprotein complex is composed of a single-guide RNA (sgRNA) that enables targeting of the Cas9 endonuclease to specific sequences in the genome, where Cas9 introduces a blunt-ended double-strand break (DSB) that then needs to be repaired65,67 (see the figure). Repair can occur through either the homologous recombination (HR) repair pathway or end-joining pathways such as non-homologous end-joining (NHEJ) and alternative end-joining (Alt-EJ). HR can be carried out using either the sister chromatid or exogenous donor DNA if it is available, although repair using donor DNA occurs at a very low rate and is not currently useful for screening. End-joining pathways result in small insertions and/or deletions (indels). These small indels are normally selected for in CRISPR screens, as normal error-free repair will re-establish the endogenous sequence, which can then be re-cut by the sgRNA-guided Cas9 to restart the cycle. Libraries of sgRNAs for screening are designed to target the open reading frames (ORFs) of the genome69–73. Indels in the ORF can result in either a frameshift that creates a knockout of the allele through nonsense-mediated decay of the mRNA or truncation of the protein or an in-frame mutation that may or may not exert a phenotypic effect depending on the structural or functional importance of the altered region70.