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High-throughput functional genomics using CRISPR–Cas9

Key Points

  • The RNA-mediated simple programmability of Cas9 opens new and exciting avenues for genome-scale functional interrogation of the genome.

  • Cas9 can be used for both nuclease-mediated gene knockout and transcriptional modulation approaches. The mechanisms of these perturbations differ substantially from the more established RNA interference (RNAi) approaches for targeted genetic screens.

  • Screening applications can be carried out in a wide range of formats using different molecular reagents and delivery vehicles. These will have an effect on the possible applications, readout and perturbation kinetics.

  • Initial Cas9-based screens displayed remarkable results: high consistency across unique reagents that target the same genetic elements, high rates of editing and large phenotypic effects.

  • There are still several challenges in the further development of Cas9-based genetic screens, such as unbiased investigation into false-negative rates, more unbiased evaluation of off-target effects, increased efficacy of designed reagents and improved readout methods.

Abstract

Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Recently, the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. In this Review, we describe recent advances using Cas9 for genome-scale screens, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. We discuss practical aspects of screen design, provide comparisons with RNA interference (RNAi) screening, and outline future applications and challenges.

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Figure 1: Molecular mechanisms underlying gene perturbation via lentiviral delivery of RNA interference reagents, Cas9 nuclease and dCas9 transcriptional effectors.
Figure 2: dCas9-mediated transcriptional modulation.
Figure 3: Screening strategies in either arrayed or pooled formats.
Figure 4: Distinct expression distributions for knockdown and knockout of a gene.

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Acknowledgements

The authors thank L. Solomon for help with illustrations, J. Wright for manuscript review and members of the Zhang Laboratory for discussions. O.S. is supported by a Klarman Family Foundation Fellowship. N.E.S. is supported by a Simons Center for the Social Brain Postdoctoral Fellowship and by the National Human Genome Research Institute (NHGRI) of the US National Institutes of Health under award number K99-HG008171. F.Z. is supported by the US National Institute of Mental Health (NIMH) (DP1-MH100706), the US National Institute of Neurological Disorders and Stroke (NINDS) (R01-NS07312401), a US National Science Foundation (NSF) Waterman Award, the Keck, Damon Runyon, Searle Scholars, Klingenstein, Vallee, Merkin, Simons, and New York Stem Cell Foundations, and Bob Metcalfe. F.Z. is a New York Stem Cell Foundation Robertson Investigator.

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Correspondence to Ophir Shalem or Feng Zhang.

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N.E.S., O.S. and F.Z. are named on patent applications related to this work. F.Z. is a cofounder of Editas Medicine and a scientific adviser for Editas Medicine and Horizon Discovery.

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Glossary

Small interfering RNA

(siRNA). RNA molecules that are 21–23 nucleotides long and that are processed from long double-stranded RNAs; they are functional components of the RNA-induced silencing complex (RISC). siRNAs typically target and silence mRNAs by binding perfectly complementary sequences in the mRNA and causing their degradation and/or translational inhibition.

Short hairpin RNA

(shRNA). Small RNAs forming hairpins that can induce sequence-specific silencing in mammalian cells through RNA interference, both when expressed endogenously and when produced exogenously and transfected into the cell.

microRNA

(miRNA). Small RNA molecules processed from hairpin-containing RNA precursors that are produced from endogenous miRNA-encoding genes. miRNAs are 21–23 nucleotides in length and, through the RNA-induced silencing complex (RISC), they target and silence mRNAs containing imperfectly complementary sequences.

Indel

(Insertion and deletion). Mutations due to small insertions or deletions of DNA sequences.

Single guide RNA

(sgRNA). An artificial fusion of CRISPR (clustered regularly interspaced short palindromic repeat) RNA (crRNA) and transactivating crRNA (tracrRNA) with critical secondary structures for loading onto Cas9 for genome editing. It functionally substitutes the complex of crRNA and tracrRNA that occurs in natural CRISPR systems. It uses RNA–DNA hybridization to guide Cas9 to the genomic target.

Nonsense-mediated decay

(NMD). An mRNA surveillance mechanism that degrades mRNAs containing nonsense mutations to prevent the expression of truncated or erroneous proteins.

CRISPRi

An engineered transcriptional silencing complex based on catalytically inactive Cas9 (dCas9) fusions and/or single guide RNA (sgRNA) modification.

CRISPRa

An engineered transcriptional activation complex based on catalytically inactive Cas9 (dCas9) fusions and/or single guide RNA (sgRNA) modification.

False-positive

Pertaining to screening results: in a screen that results in a set of putative gene hits associated with a phenotype, a false positive is a gene that is predicted to be associated but that is actually not associated with the phenotype.

False-negative

Pertaining to screening results: in a screen that results in a set of putative gene hits associated with a phenotype, a false negative is a true hit that was missed.

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Shalem, O., Sanjana, N. & Zhang, F. High-throughput functional genomics using CRISPR–Cas9. Nat Rev Genet 16, 299–311 (2015). https://doi.org/10.1038/nrg3899

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