Enhanced Cas12a multi-gene regulation using a CRISPR array separator

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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.

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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

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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)..

"This ORCID iD identifies the author of this article:" 0000-0002-3928-8959
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)..

"This ORCID iD identifies the author of this article:" 0000-0002-6717-2267
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)..
Lei S Qi

Bioengineering Department, Stanford University, Stanford, United States

For correspondence
stanley.qi@stanford.edu

Competing interests
Lei S Qi, The authors have filed a US provisional patent application related to this work (application no. 63/139,095)..

"This ORCID iD identifies the author of this article:" 0000-0002-3965-3223

Funding

Li Ka Shing Foundation

Lei S Qi

NIH Common Fund 4D Nucleome Program (U01 EB021240)

Lei S 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.

Copyright

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.