Multiplexed genome regulation in vivo with hyper-efficient Cas12a.
Lucie Y GuoJing BianAlexander E DavisPingting LiuHannah R KemptonXiaowei ZhangAugustine ChemparathyBaokun GuXueqiu LinDraven A RaneXiaoshu XuRyan M JamiolkowskiYang HuSui WangLei S QiPublished in: Nature cell biology (2022)
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.
Keyphrases
- gene therapy
- genome wide
- crispr cas
- genome editing
- wild type
- copy number
- genome wide identification
- single cell
- dna methylation
- diabetic retinopathy
- transcription factor
- optic nerve
- optical coherence tomography
- rna seq
- south africa
- stem cells
- genome wide analysis
- high throughput
- cell therapy
- signaling pathway
- high resolution
- protein protein
- nuclear factor
- gene expression
- inflammatory response
- mass spectrometry
- high fat diet induced
- metabolic syndrome
- amino acid
- type diabetes
- immune response
- bioinformatics analysis
- skeletal muscle