Enhanced Cas12a multi-gene regulation using a CRISPR array separator
- Stanford University, United States
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
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.
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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