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Rational designs of Cas12a nuclease with the improved targeting specificity at both the proximal and distal region from the PAM

ABSTRACT: Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting at the distal region from the PAM.

ORGANISM(S): Homo sapiens

PROVIDER: GSE229888 | GEO | 2023/04/17

REPOSITORIES: GEO

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Randomized PAM depletion of AsCas12a

Project description:We report the PAMs of AsCas12a using a cell-free TXTL-based cleavage assay. By adding randomized PAM library and AsCas12a-gRNA in vitro, functional PAM sequences were cleaved, while non-functional PAMs remained. By amplifying the non-cleaved DNA, we use next-generation sequencing to analyze the depletion of functional PAMs of AsCas12a.

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Project description:We report the PAMs of phylogenetically-diverse Cas12a nucleases using a cell-free TXTL-based PAM screen. By adding a 5N randomized PAM library and Cas12a-gRNA in vitro, recognized PAM sequences were cleaved, while non-recognized PAMs remained. By amplifying the non-cleaved DNA, we used next-generation sequencing to analyze the depletion of functional PAMs of these Cas12a nucleases.

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Project description:We report the PAMs of NmeCas9 using a cell-free TXTL-based cleavage assay. By adding randomized PAM library and NmeCas9-gRNA in vitro, functional PAM sequences were cleaved, while non-functional PAMs remained. By amplifying the non-cleaved DNA, we use next-generation sequencing to analyze the depletion of functional PAMs of NmeCas9.

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seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression [PAM-1 ChIRP-seq]

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seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression [PAM-1 KO ChIP-seq]

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| S-EPMC7144938 | biostudies-literature

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