2014
DOI: 10.1126/science.1247005
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Abstract: The simplicity of programming the CRISPR-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a… Show more

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Cited by 4,223 publications
(4,039 citation statements)
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References 27 publications
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“…Future work will be focused on the use of a suitable control data set to compare this analysis pipeline with other approaches such as shRNAseq 5 . We anticipate that the approach for differential representation analysis described in this paper will also be useful in the analysis of short-guided RNA-seq (sgRNA-seq) knockout screens as facilitated by the clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) system 18, 19 .…”
Section: Discussionmentioning
confidence: 99%
“…Future work will be focused on the use of a suitable control data set to compare this analysis pipeline with other approaches such as shRNAseq 5 . We anticipate that the approach for differential representation analysis described in this paper will also be useful in the analysis of short-guided RNA-seq (sgRNA-seq) knockout screens as facilitated by the clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) system 18, 19 .…”
Section: Discussionmentioning
confidence: 99%
“…However, copynumber variation, including large duplications or deletions, can also profoundly influence health. The development of Cas9-mediated genome-wide gene knockout 75,76 and overexpression 77 screening highlights how multiplex assays could be used for understanding the effects of copy-number variation. These assays quantify the effect of single-gene deletion or overexpression.…”
Section: Limitations Of Maves and How To Overcome Themmentioning
confidence: 99%
“…Lentivirus CRISPR constructs targeting hCYR61, rSmad2 and rSmad3 were made using the LentiCRISPRv2 vector (Addgene Plasmid 52961) following the GeCKO protocol (24,25). Briefly, the lentiCRISPRv2 vector was digested by BsmB1 and de-phosphorylated by CIP alkaline phosphatase.…”
Section: Lentivirusmentioning
confidence: 99%
“…Briefly, the lentiCRISPRv2 vector was digested by BsmB1 and de-phosphorylated by CIP alkaline phosphatase. sgRNA target sequences were designed using the GeCKO library (24,25) (sequences listed in Supplementary Table S1, available at Carcinogenesis Online), and the synthesized oligos were annealed and phosphorylated using T4 polynucleotide kinase. The annealed sgRNA target sequence oligos were ligated into the digested lentiCRISPRv2 backbone using T4 DNA ligase.…”
Section: Lentivirusmentioning
confidence: 99%