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      Systematic comparison of CRISPR-Cas9 and RNAi screens for essential genes

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          Abstract

          We compare the ability of shRNA and CRISPR/Cas9 screens to identify essential genes in the human chronic myelogenous leukemia cell line K562. We find that the precision of the two libraries in detecting essential genes is similar and that combining data from both screens improves performance. Notably, results from the two screens show little correlation, which can be partially explained by identification of distinct essential biological processes with each technology.

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          Expression profiling reveals off-target gene regulation by RNAi.

          Aimee Jackson, Steven Bartz, Janell M Schelter … (2003)
          RNA interference is thought to require near-identity between the small interfering RNA (siRNA) and its cognate mRNA. Here, we used gene expression profiling to characterize the specificity of gene silencing by siRNAs in cultured human cells. Transcript profiles revealed siRNA-specific rather than target-specific signatures, including direct silencing of nontargeted genes containing as few as eleven contiguous nucleotides of identity to the siRNA. These results demonstrate that siRNAs may cross-react with targets of limited sequence similarity.
          Article 0 comments Cited 529 times – based on 0 reviews     Review now
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            Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.

            Hiroko Koike-Yusa, Yilong Li, E-Pien Tan … (2014)
            Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.
            Article 0 comments Cited 453 times – based on 0 reviews     Review now
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              Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains

              Junwei Shi, Eric Wang, Joseph Milazzo … (2015)
              CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-induced mutations to the 5’ exons of candidate genes 1–5 , but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. We show that the magnitude of negative selection reports the functional importance of individual protein domains of interest. A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 9604648
                Journal ID (pubmed-jr-id): 20305
                Journal ID (nlm-ta): Nat Biotechnol
                Journal ID (iso-abbrev): Nat. Biotechnol.
                Title: Nature biotechnology
                ISSN (Print): 1087-0156
                ISSN (Electronic): 1546-1696
                Publication date Nihms-submitted: 1 May 2016
                Publication date (Electronic): 09 May 2016
                Publication date (Print): June 2016
                Publication date PMC-release: 09 November 2016
                Volume: 34
                Issue: 6
                Pages: 634-636
                Affiliations
                [1 ]Department of Genetics, Stanford University, Stanford, CA, USA
                [2 ]Department of Chemistry, Stanford University, Stanford, CA, USA
                [3 ]Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, CA, USA
                Author notes
                [4 ]Corresponding Author: bassik@ 123456stanford.edu
                Article
                Manuscript ID: NIHMS777404
                DOI: 10.1038/nbt.3567
                PMC ID: 4900911
                PubMed ID: 27159373
                SO-VID: 4a61eb74-4618-4075-beba-99a006c5e7b0
                License:

                Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

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                ScienceOpen disciplines: Biotechnology
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                ScienceOpen disciplines: Biotechnology

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