Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based technology enables efficient and precise perturbations of target genomic sites. Combining the endonuclease Cas9 and a pooled guide RNA library allows for systematic screenings of genes associated with a growth disadvantage or lethal phenotype under various conditions in organisms and tissues. Here, we describe a complete protocol for scalable CRISPR/Cas9-based dropout screening for essential genes from focused genomic regions to whole genomes.
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References
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://doi.org/10.1126/science.1231143
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918. https://doi.org/10.1016/j.cell.2013.04.025
Rouet P, Smih F, Jasin M (1994) Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol 14(12):8096–8106
Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32(12):1262–1267. https://doi.org/10.1038/nbt.3026
Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343(6166):80–84. https://doi.org/10.1126/science.1246981
Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343(6166):84–87. https://doi.org/10.1126/science.1247005
Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera MC, Yusa K (2014) Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32(3):267–273. https://doi.org/10.1038/nbt.2800
Shalem O, Sanjana NE, Zhang F (2015) High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet 16(5):299–311. https://doi.org/10.1038/nrg3899
Wang T, Birsoy K, Hughes NW, Krupczak KM, Post Y, Wei JJ, Lander ES, Sabatini DM (2015) Identification and characterization of essential genes in the human genome. Science 350(6264):1096–1101. https://doi.org/10.1126/science.aac7041
Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8):783–784. https://doi.org/10.1038/nmeth.3047
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832. https://doi.org/10.1038/nbt.2647
Li W, Köster J, Xu H, Chen CH, Xiao T, Liu JS, Brown M, Liu XS (2015) Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR. Genome Biol 16:281. https://doi.org/10.1186/s13059-015-0843-6
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Wu, K., Malek, S.N. (2019). CRISPR/Cas9-Based Gene Dropout Screens. In: Malek, S. (eds) Chronic Lymphocytic Leukemia. Methods in Molecular Biology, vol 1881. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8876-1_15
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DOI: https://doi.org/10.1007/978-1-4939-8876-1_15
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