Enhanced Cas12a multi-gene regulation using a CRISPR array separator

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 hypersensiti...

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Autores principales:	Jens P Magnusson, Antonio Ray Rios, Lingling Wu, Lei S Qi
Formato:	article
Lenguaje:	EN
Publicado:	eLife Sciences Publications Ltd 2021
Materias:	CRISPR Cas12a multiplexed gene regulation CRISPR array separator GC content Medicine R Science Q Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/998351ecbc984fe692f16e379a5ef185
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id	oai:doaj.org-article:998351ecbc984fe692f16e379a5ef185
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spelling	oai:doaj.org-article:998351ecbc984fe692f16e379a5ef1852021-11-15T06:43:14ZEnhanced Cas12a multi-gene regulation using a CRISPR array separator10.7554/eLife.664062050-084Xe66406https://doaj.org/article/998351ecbc984fe692f16e379a5ef1852021-09-01T00:00:00Zhttps://elifesciences.org/articles/66406https://doaj.org/toc/2050-084XThe 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.Jens P MagnussonAntonio Ray RiosLingling WuLei S QieLife Sciences Publications LtdarticleCRISPRCas12amultiplexed gene regulationCRISPR arrayseparatorGC contentMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR Cas12a multiplexed gene regulation CRISPR array separator GC content Medicine R Science Q Biology (General) QH301-705.5
spellingShingle	CRISPR Cas12a multiplexed gene regulation CRISPR array separator GC content Medicine R Science Q Biology (General) QH301-705.5 Jens P Magnusson Antonio Ray Rios Lingling Wu Lei S Qi Enhanced Cas12a multi-gene regulation using a CRISPR array separator
description	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.
format	article
author	Jens P Magnusson Antonio Ray Rios Lingling Wu Lei S Qi
author_facet	Jens P Magnusson Antonio Ray Rios Lingling Wu Lei S Qi
author_sort	Jens P Magnusson
title	Enhanced Cas12a multi-gene regulation using a CRISPR array separator
title_short	Enhanced Cas12a multi-gene regulation using a CRISPR array separator
title_full	Enhanced Cas12a multi-gene regulation using a CRISPR array separator
title_fullStr	Enhanced Cas12a multi-gene regulation using a CRISPR array separator
title_full_unstemmed	Enhanced Cas12a multi-gene regulation using a CRISPR array separator
title_sort	enhanced cas12a multi-gene regulation using a crispr array separator
publisher	eLife Sciences Publications Ltd
publishDate	2021
url	https://doaj.org/article/998351ecbc984fe692f16e379a5ef185
work_keys_str_mv	AT jenspmagnusson enhancedcas12amultigeneregulationusingacrisprarrayseparator AT antoniorayrios enhancedcas12amultigeneregulationusingacrisprarrayseparator AT linglingwu enhancedcas12amultigeneregulationusingacrisprarrayseparator AT leisqi enhancedcas12amultigeneregulationusingacrisprarrayseparator
_version_	1718428568278532096

Enhanced Cas12a multi-gene regulation using a CRISPR array separator

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