Multiplex CRISPR genome regulation in mouse retina with hyper-efficient Cas12a

Guo, Lucie Y.; Bian, Jing; Davis, A. Edward; Liu, Pingting; Kempton, H.; Zhang, Xiaowei; Chemparathy, Augustine; Gu, Baokun; Lin, Xueqiu; Rane, Draven A.; Jamiolkowski, Ryan M.; Hu, Yang; Wang, Sui; Qi, Lei S.

doi:10.21203/rs.3.pex-1811/v1

Cited by 3 publications

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“…Nuclease-dead dCas12a from Lachnospiraceae bacterium and its crRNA backbone were modified from previous publication 24 . The step-by-step protocol for generating crRNA arrays is provided at Nature Protocol Exchange 53 .…”

Section: Methodsmentioning

confidence: 99%

“…More than 350,000 reads were generated with each sample using Illumina platform. Data analysis was performed with CRISPResso2 53 .…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid DNA was injected into the subretinal space of neonatal mice, and electrical pulses were applied with tweezer-style electrodes as described 37 , 38 . More details are provided at Nature Protocol Exchange 53 . Plasmid with wildtype dCas12a was mixed with CAG-GFP construct in ~5:1 ratio and electroporated at a concentration of up to 2μg/μl total plasmid at P0.…”

Section: Methodsmentioning

confidence: 99%

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Multiplexed genome regulation in vivo with hyper-efficient Cas12a

et al. 2022

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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 crRNAs from a single transcript, however, their low efficiency has hindered applications in vivo . Through structure-guided protein engineering, we develop a hyper-efficient LbCas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low crRNA conditions. We demonstrate that hyperdCas12a has minimal off-target effects compared to the wildtype system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a-activator and a single crRNA array simultaneously activating endogenous Oct4, Sox2, and Klf4 genes in the retina of postnatal 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.

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Section: Methodsmentioning

confidence: 99%

“…More than 350,000 reads were generated with each sample using Illumina platform. Data analysis was performed with CRISPResso2 53 .…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Multiplexed genome regulation in vivo with hyper-efficient Cas12a

et al. 2022

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“…Several groups have reported an engineering strategy that involves introducing cationic residues to enhance target DNA binding, thereby improving the function of CRISPR-Cas or relevant proteins [36][37][38] .…”

Section: Discussionmentioning

confidence: 99%

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Eggers,

Chen,

Soczek

et al. 2023

Preprint

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SUMMARYThermostable CRISPR-Cas9 enzymes could improve genome editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold higher genome editing levels. Cryo-EM structures of the wildtype and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome editing activity.

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“…Such a design allowed to reach ~9,000 dCas9 molecules per E. coli cell compared to ~500 in the wild-type case. Some other examples of dCas engineering include altering the PAM interacting domain of SpdCas9 to specifically recognize start codons of proteincoding genes (5′-CAT-3′ PAM) (Wang et al, 2021) and development of hyperdCas12a-a version of dCas12a from Lachnospiraceae bacterium with enhanced binding affinity resulting in significantly improved CRISPRi and CRISPRa outcomes in eukaryotes (Guo et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems

Shaytan

Novikov

Vinnikov

et al. 2022

Front. Mol. Biosci.

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In the last decade, the CRISPR-Cas technology has gained widespread popularity in different fields from genome editing and detecting specific DNA/RNA sequences to gene expression control. At the heart of this technology is the ability of CRISPR-Cas complexes to be programmed for targeting particular DNA loci, even when using catalytically inactive dCas-proteins. The repertoire of naturally derived and engineered dCas-proteins including fusion proteins presents a promising toolbox that can be used to construct functional synthetic genetic circuits. Rational genetic circuit design, apart from having practical relevance, is an important step towards a deeper quantitative understanding of the basic principles governing gene expression regulation and functioning of living organisms. In this minireview, we provide a succinct overview of the application of CRISPR-dCas-based systems in the emerging field of synthetic genetic circuit design. We discuss the diversity of dCas-based tools, their properties, and their application in different types of genetic circuits and outline challenges and further research directions in the field.

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Multiplex CRISPR genome regulation in mouse retina with hyper-efficient Cas12a

Cited by 3 publications

References 0 publications

Multiplexed genome regulation in vivo with hyper-efficient Cas12a

Multiplexed genome regulation in vivo with hyper-efficient Cas12a

Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems

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