Abstract
Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides. Here, we describe a method for constructing complex plasmid libraries based on pooled designed oligomers with high representation and tight distributions. The procedure allows construction of plasmid libraries of >60,000 members with a 95th/5th percentile ratio of less than 3.5.
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References
Chang K, Elledge SJ, Hannon GJ (2006) Lessons from nature: microRNA-based shRNA libraries. Nat Methods 3:707–714
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
Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman JS (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159(3):647–661
Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F (2015) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517(7536):583–588
Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ovanesyan DT, Qian J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJK, Ebrahimkhani MR, Collins JJ, Weiss R, Church G (2015) Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods 12(11):1051–1054
Morgens DW, Deans RM, Li A, Bassik MC (2016) Systematic comparison of CRISPR-Cas9 and RNAi screens for essential genes. Nat Biotechnol 34(6):634–636
Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8):783–784
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Carstens, C.P., Felts, K.A., Johns, S.E. (2018). Construction of CRISPR Libraries for Functional Screening. In: Braman, J. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1772. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7795-6_7
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DOI: https://doi.org/10.1007/978-1-4939-7795-6_7
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Online ISBN: 978-1-4939-7795-6
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