Abstract

The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data have been provided for all figures.

Article and author information

Author details

  1. Jens P Magnusson

    Bioengineering Department, Stanford University, Stanford, United States
    Competing interests
    Jens P Magnusson, The authors have filed a US provisional patent application related to this work (application no. 63/139,095)..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3928-8959
  2. Antonio Ray Rios

    Bioengineering Department, Stanford University, Stanford, United States
    Competing interests
    Antonio Ray Rios, The authors have filed a US provisional patent application related to this work (application no. 63/139,095)..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6717-2267
  3. Lingling Wu

    Bioengineering Department, Stanford University, Stanford, United States
    Competing interests
    Lingling Wu, The authors have filed a US provisional patent application related to this work (application no. 63/139,095)..
  4. Lei Stanley Qi

    Bioengineering Department, Stanford University, Stanford, United States
    For correspondence
    stanley.qi@stanford.edu
    Competing interests
    Lei Stanley Qi, The authors have filed a US provisional patent application related to this work (application no. 63/139,095)..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3965-3223

Funding

Li Ka Shing Foundation

  • Lei Stanley Qi

NIH Common Fund 4D Nucleome Program (U01 EB021240)

  • Lei Stanley Qi

Human Frontier Science Program (Long-term Fellowship)

  • Jens P Magnusson

Sweden-America Foundation

  • Jens P Magnusson

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Joseph T Wade, Wadsworth Center, New York State Department of Health, United States

Version history

  1. Received: January 9, 2021
  2. Preprint posted: January 27, 2021 (view preprint)
  3. Accepted: September 8, 2021
  4. Accepted Manuscript published: September 9, 2021 (version 1)
  5. Accepted Manuscript updated: September 13, 2021 (version 2)
  6. Version of Record published: September 28, 2021 (version 3)

Copyright

© 2021, Magnusson et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jens P Magnusson
  2. Antonio Ray Rios
  3. Lingling Wu
  4. Lei Stanley Qi
(2021)
Enhanced Cas12a multi-gene regulation using a CRISPR array separator
eLife 10:e66406.
https://doi.org/10.7554/eLife.66406

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https://doi.org/10.7554/eLife.66406