Genetic screens in human cells using the CRISPR-Cas9 system
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Improving Whole-Genome Screens: Improved methods are needed for the knockout of individual genes in genome-scale functional screens. Wang et al. (p. 80 , published online 12 December) and Shalem et al. (p. 84 , published online 12 December) used the bacterial CRISPR/Cas9 system to power-screen protocols that avoid several of the pitfalls associated with small interfering RNA (siRNA) screens. Genome editing by these methods completely disrupts target genes, thus avoiding weak signals that can occur when transcript abundance is partially decreased by siRNA. Furthermore, gene targeting by the CRISPR system is more precise and appears to produce substantially fewer off-target effects than existing methods.
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gRNA-Cas9 nuclease-induced DSBs repaired by NHEJ resulted in 1-10 bp deletions or 1 bp insertions in as many as 90% of cells.
“The gRNA–Cas9 nuclease complex directed double-stranded breaks (DSBs) at target sites, which after repair by error-prone non-homologous end-joining (NHEJ)196,197, resulted in 1–10 bp deletions or 1 bp insertions (‘indels’) in as many as 90% of cells192,194 (Figure 2a).”
From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [194]
The paper reports that Cas9-induced double-strand breaks produced small deletions (<20 bp) and less frequent tiny insertions or substitutions (<3 bp) in KBM7 cells (passage [1], section labeled Abstract; page unavailable). It also reports 97% cleavage at the targeted AAVS1 locus (passage [4], same section), but does not establish the claimed 1–10 bp deletions or 1 bp insertions in as many as 90% of cells. The sentence cites other sources for the NHEJ mechanism. (LLM, conf=0.86)
Experimental — accuracy not yet measuredHistorically pooled screens rely on enriching populations for a phenotype followed by NGS to measure perturbation abundance.
“Historically, pooled screens have relied on enriching populations of cells for a phenotype of interest, followed by next-generation sequencing (NGS) to measure changes in perturbation abundance.”
From Optical Pooled Screens in Human Cells (2019) · cites this paper as Wang et al., 2014
Genome-scale pooled CRISPR loss-of-function screens allow forward genetic studies in many mammalian cell types, including primary T cells, where they have identified genes that may be targeted in future immunotherapies.
“Genome-scale pooled CRISPR loss-of-function screens now allow forward genetic studies in many mammalian cell types (Wang et al., 2014; Shalem et al., 2014) including primary T cells where they have identified genes that may be targeted in future immunotherapies (Dong et al., 2019; Shifrut et al., 2018; Wei et al., 2019).”
From Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies (2020) · cites this paper as Wang et al., 2014
DNA sequence recognition by CRISPR/Cas9 requires only a single 20-nt guide RNA and a protospacer adjacent motif, enabling rapid engineering and testing of many DNA cleavage sites.
“DNA sequence recognition requires only a single 20-nt guide RNA and a protospacer adjacent motif, which enables one to rapidly engineer and test a large number of DNA cleavage sites (17)(18)(19)(20).”
From RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection (2014) · cites this paper as [18]
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- Authors
- Wang, T., Wei, J. J., Sabatini, D. M., & Lander, E. S.
- Affiliations
- Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
- Whitehead Institute for Biomedical Research (US)
- Broad Institute (US)
- Corresponding author
- Sabatini, David M.
- Lander, Eric S.
- Venue
- Science
- Published
- 2014-01-03
- Volume
- 343
- Issue
- 6166
- Pages
- 80-84
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- PMC
- PMC3972032
- OpenAlex
- W1594078235
- Cited by
- 2,856
- References
- 43
- Citations by year
- 2026: 115 · 2025: 142 · 2024: 181 · 2023: 168 · 2022: 208 · 2021: 240 · 2020: 270 · 2019: 277 · 2018: 266 · 2017: 315 · 2016: 271 · 2015: 226 · 2014: 168 · 2013: 5
- FWCI
- 100.11
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Science (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- Funders
- National Science Foundation
- Broad Institute
- National Institutes of Health
- National Human Genome Research Institute (U54HG003067)
- National Cancer Institute (CA103866, R01CA103866)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- CRISPR/Cas9, single guide RNA library, loss-of-function screening, positive and negative selection, 6-thioguanine resistance, DNA mismatch repair, etoposide sensitivity, TOP2A, CDK6, essential genes, sequence motifs, human cell lines, isogenic cell lines
- MeSH terms
- Poly-ADP-Ribose Binding Proteins, Antigens, Neoplasm, Antimetabolites, Antineoplastic, Antineoplastic Agents, Phytogenic, DNA Topoisomerases, Type II, DNA-Binding Proteins, Etoposide, Genetic Testing, Humans, RNA, Thioguanine, Gene Library, Drug Resistance, Neoplasm, DNA Mismatch Repair, Genome-Wide Association Study, Gene Knockout Techniques, Clustered Regularly Interspaced Short Palindromic Repeats
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