Massively parallel Cas13 screens reveal principles for guide RNA design
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Abstract
Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knockdown without altering the genome. To define rules for the design of Cas13d guide RNAs (gRNAs), we conducted massively parallel screens targeting messenger RNAs (mRNAs) of a green fluorescent protein transgene, and CD46, CD55 and CD71 cell-surface proteins in human cells. In total, we measured the activity of 24,460 gRNAs with and without mismatches relative to the target sequences. Knockdown efficacy is driven by gRNA-specific features and target site context. Single mismatches generally reduce knockdown to a modest degree, but spacer nucleotides 15-21 are largely intolerant of target site mismatches. We developed a computational model to identify optimal gRNAs and confirm their generalizability, testing 3,979 guides targeting mRNAs of 48 endogenous genes. We show that Cas13 can be used in forward transcriptomic pooled screens and, using our model, predict optimized Cas13 gRNAs for all protein-coding transcripts in the human genome.
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The authors previously confirmed that RfxCas13d can cause strong target-RNA knockdown and derived optimal targeting rules from thousands of guide RNAs tiling multiple transcripts.
“Recently, we confirmed that RfxCas13d can lead to striking target-RNA knockdown, and learned a set of optimal targeting rules from thousands of guide RNAs tiling multiple transcripts11.”
From Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq (2023) · cites this paper as [11]
Log2FC enrichments were generally more pronounced in samples using the enhanced direct repeat.
“As we have previously described11, we noticed that log2FC enrichments were generally more pronounced in samples using the enhanced direct repeat.”
From Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq (2023) · cites this paper as [11]
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- Authors
- Wessels, H.-H., Méndez-Mancilla, A., Guo, X., Legut, M., Daniloski, Z., & Sanjana, N. E.
- Affiliations
- New York Genome Center (US)
- Corresponding author
- Sanjana, Neville E.
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2020-03-16
- Volume
- 38
- Issue
- 6
- Pages
- 722-727
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC7294996
- OpenAlex
- W3011119456
- Cited by
- 418
- References
- 48
- Citations by year
- 2026: 64 · 2025: 72 · 2024: 86 · 2023: 50 · 2022: 72 · 2021: 49 · 2020: 25
- FWCI
- 15.96
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- Nature Biotechnology (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (R00HG008171, DP2HG010099)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- Cas13, guide RNA design, mRNA knockdown
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Humans, Sequence Analysis, RNA, Computational Biology, Gene Knockdown Techniques, HEK293 Cells, CRISPR-Cas Systems
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