Multiplexed genome regulation in vivo with hyper-efficient Cas12a

Lucie Y Guo; Jing Bian; Alexander E Davis; Pingting Liu; Hannah R Kempton; Xiaowei Zhang; Augustine Chemparathy; Baokun Gu; Xueqiu Lin; Draven A Rane; Xiaoshu Xu; Ryan M Jamiolkowski; Yang Hu; Sui Wang; Lei S Qi

doi:10.1038/s41556-022-00870-7

Multiplexed genome regulation in vivo with hyper-efficient Cas12a

Nat Cell Biol. 2022 Apr;24(4):590-600. doi: 10.1038/s41556-022-00870-7. Epub 2022 Apr 12.

Authors

Lucie Y Guo^#^{1

2}, Jing Bian^#³, Alexander E Davis⁴, Pingting Liu⁴, Hannah R Kempton³, Xiaowei Zhang³, Augustine Chemparathy³, Baokun Gu³, Xueqiu Lin³, Draven A Rane³, Xiaoshu Xu³, Ryan M Jamiolkowski³, Yang Hu⁴, Sui Wang⁵, Lei S Qi^{6

7

8}

Affiliations

¹ Department of Bioengineering, Stanford University, Stanford, CA, USA. lucieguo@stanford.edu.
² Department of Ophthalmology, Stanford University, Stanford, CA, USA. lucieguo@stanford.edu.
³ Department of Bioengineering, Stanford University, Stanford, CA, USA.
⁴ Department of Ophthalmology, Stanford University, Stanford, CA, USA.
⁵ Department of Ophthalmology, Stanford University, Stanford, CA, USA. suiwang@stanford.edu.
⁶ Department of Bioengineering, Stanford University, Stanford, CA, USA. stanley.qi@stanford.edu.
⁷ Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA. stanley.qi@stanford.edu.
⁸ Stanford ChEM-H Institute, Stanford University, Stanford, CA, USA. stanley.qi@stanford.edu.

^# Contributed equally.

Abstract

Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Associated Proteins* / genetics
CRISPR-Associated Proteins* / metabolism
CRISPR-Cas Systems
Gene Editing
Mice
RNA / metabolism

Substances

CRISPR-Associated Proteins
RNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding