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Am I ready for CRISPR? A user's guide to genetic screens

Key Points

  • Pooled screens are a cost-effective approach to investigating phenotypes at the genome scale and build on technological innovations with lentivirus, oligonucleotide synthesis and massively parallel sequencing.

  • CRISPR technology is extremely powerful, with many modalities for perturbing gene function. A successful genetic screen, however, requires not only a good library of perturbations but also a relevant model and optimized assay.

  • When executing a screen, maintaining representation of the library is essential to good quantification. All steps in the process should be investigated experimentally in advance of the screen to ensure efficiency.

  • Designing a library of single-guide RNAs (sgRNAs) requires genomic information from multiple sources, which will change over time as the genome is better annotated and may vary depending on the type of cell under investigation.

  • Defining a systematic follow-up path, both analytically and experimentally, should be thought through before conducting a screen. Secondary pooled screens with customized libraries can be very powerful at this stage.

Abstract

Exciting new technologies are often self-limiting in their rollout, as access to state-of-the-art instrumentation or the need for years of hands-on experience, for better or worse, ensures slow adoption by the community. CRISPR technology, however, presents the opposite dilemma, where the simplicity of the system enabled the parallel development of many applications, improvements and derivatives, and new users are now presented with an almost paralyzing abundance of choices. This Review intends to guide users through the process of applying CRISPR technology to their biological problems of interest, especially in the context of discovering gene function at scale.

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Figure 2: Diversity of activities with Cas9.
Figure 1: Maintaining representation in pooled screens.
Figure 3: Design considerations for CRISPR-based knockout.

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Acknowledgements

The author thanks K. Donovan for assistance in manuscript preparation, M. Hegde for assistance with analysis and the entire Genetic Perturbation Platform (GPP) at the Broad Institute. For insightful discussions, the author thanks J. Arroyo, O. Parnas and Z. Tothova (Broad Institute); J. Listgarten and N. Fusi (Microsoft Research); C.Wilen and R. Orchard (Washington University); J. Klappenbach (Merck); L. Brody (Desktop Genetics); and M. Fan (Addgene). This work is dedicated to Francis Edward Sheehan.

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Correspondence to John G. Doench.

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J.G.D. is a consultant for Tango Therapeutics.

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Glossary

Confirmation bias

The tendency to focus on information that confirms a pre-existing belief to the exclusion of contradictory information. In genetic screens, this can manifest in choosing to follow up a gene that scores with marginal statistical significance in the primary screen, rather than focusing on the experimentally identified top hits.

Single-guide RNAs

(sgRNAs). The first CRISPR systems characterized in prokaryotes required two RNAs to program the Cas9 protein: a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). To simplify the system, these two independent RNAs can instead be merged into a single transcript, the sgRNA, which has practical benefits especially for ease of expression in mammalian cells.

Titre

The titre of a lentivirus is the number of infectious particles per unit of volume, and the ratio of lentiviral integrants to cells is the multiplicity of infection (MOI). Importantly, cells differ in their inherent infectivity, and thus the volume of virus that is sufficient to achieve a given infection efficiency in cell type A is not necessarily the same in cell type B.

Xenograft

The transplantation of cells from one species to another. Often, it involves introducing human cancer cells into a mouse model to study their behaviour in complex microenvironments that are difficult to model in cell culture. Mice with an active immune system will recognize foreign cells and clear them out; thus, such experiments must be performed in immunodeficient mice.

Paralogues

Two genes that are produced by a gene duplication event and that, owing to their shared sequence, may have the same or similar functions. Thus, loss of one of them is often insufficient to manifest a phenotype, as the other paralogue can compensate.

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Doench, J. Am I ready for CRISPR? A user's guide to genetic screens. Nat Rev Genet 19, 67–80 (2018). https://doi.org/10.1038/nrg.2017.97

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