Identifying synthetic lethal targets using CRISPR/Cas9 system

• Published in: Methods (San Diego, Calif.) Calif.), 2017-12, Vol.131, p.66-73
• Main Authors: Dhanjal, Jaspreet Kaur, Radhakrishnan, Navaneethan, Sundar, Durai
• Format: Article
• Language: English
• Subjects: Arrayed screen; Computational Biology - methods; CRISPR-Cas Systems - genetics; CRISPR/Cas9; DNA Repair - genetics; Endonucleases - genetics; Gene Knockdown Techniques - methods; Gene Library; Genetic Engineering - methods; Genetic Therapy - methods; High-Throughput Nucleotide Sequencing; High-Throughput Screening Assays - methods; Humans; Knockout library; Loss of Function Mutation - genetics; Neoplasms - genetics; Neoplasms - therapy; Pooled screen; RNA Interference; RNA, Small Interfering - genetics; Sequence Analysis, DNA; Small Molecule Libraries; Synthetic Lethal Mutations - genetics; Synthetic lethality
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2017.07.007

•Synthetic lethal target interactions are paving way for new cancer therapies.•CRISPR/Cas9 is the most recent technology for identification of synthetic lethal targets.•sgRNA knockout libraries are commercially available for creating loss of function mutant cell lines.•Complete knockdown of genes makes CRISPRs advantageous over other RNAi approaches. Synthetic lethality occurs when co-occurrence of two genetic events is unfavorable for the survival of the cell or organism. The conventional approach of high throughput screening of synthetic lethal targets using chemical compounds has been replaced by RNAi technology. CRISPR/Cas9, an RNA guided endonuclease system is the most recent technology for this work. Here, we have discussed the major considerations involved in designing a CRISPR/Cas9 based screening experiment for identification of synthetic lethal targets. It mainly includes CRISPR library to be used, cell types for conducting the experiment, the most appropriate screening strategy and ways of selecting the desired phenotypes from the complete cell population. The complete knockdown of genes can be achieved using CRISPR/Cas9 knockout libraries. For higher quality loss-of-function screens, haploid cells with defective homology-directed DNA repair mechanism could be used. Two widely used screening formats include arrayed and pooled screens followed by negative or positive selection of the cells with desired phenotype. However, pooled screening format with negative selection of cells serves the best. The advantages of using CRISPR/Cas9 system over the other RNAi approaches have also been discussed. Finally, some studies using CRISPR/Cas9 for genome-wide knockout screening in human cells and computational approaches for identification of synthetic lethal interactions have been discussed.