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Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition

ABSTRACT: our findings show that the Cas proteins could be designed to be stringent in PAM recognition to reduce their off-target effects , which points out a promising and practical way for the minimization of the off-targeting in genome editing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE185486 | GEO | 2021/10/09

REPOSITORIES: GEO

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Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition

Project description:Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition

| PRJNA769264 | ENA

A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

Project description:Our study shows that CeCas12a nuclease is active in human cells and the stringency of PAM recognition could be an important factor shaping off-target editing in gene editing. Thus, CeCas12a provides a promising candidate with distinctive characteristics for research and therapeutic applications

2020-03-06 | GSE146420 | GEO

Randomized PAM depletion of phylogenetically-diverse Cas12a nucleases

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.

2020-03-02 | GSE130377 | GEO

A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing.

Project description:BackgroundAsCas12a and LbCas12a nucleases are reported to be promising tools for genome engineering with protospacer adjacent motif (PAM) TTTV as the optimal. However, the C-containing PAM (CTTV, TCTV, TTCV, etc.) recognition by Cas12a might induce extra off-target edits at these non-canonical PAM sites.ResultsHere, we identify a novel Cas12a nuclease CeCas12a from Coprococcus eutactus, which is a programmable nuclease with genome-editing efficiencies comparable to AsCas12a and LbCas12a in human cells. Moreover, CeCas12a is revealed to be more stringent for PAM recognition in vitro and in vivo followed by very low off-target editing rates in cells. Notably, CeCas12a renders less off-target edits located at C-containing PAM at multiple sites compared to LbCas12a and AsCas12a, as assessed by targeted sequencing methods.ConclusionsOur study shows that CeCas12a nuclease is active in human cells and the stringency of PAM recognition could be an important factor shaping off-target editing in gene editing. Thus, CeCas12a provides a promising candidate with distinctive characteristics for research and therapeutic applications.

| S-EPMC7093978 | biostudies-literature

Deep sequencing data from PAM screen of Pa83 type IV-A CRISPR-Cas system

Project description:In this work the favorable PAM sequences for a type IV-A CRISPR-Cas system were determined

2022-08-19 | GSE211446 | GEO

Vibrio Cholerae CRISPR-cas PAM requirements

Project description:Plasmids were constructed harboring protospacers that are perfect targets for spacers #4 and #21 of the array, but contain NNN (all possible PAM combinations) immediately upstream of the protospacer. The plasmids were introduced into V. cholerae with or without a functional CRISPR-cas system and cells were plated on selective media. Cells were collected and the protospacer plasmids were sequenced in a high throughput manner. PAMs were counted using a custom python script

2024-05-06 | E-MTAB-14044 | biostudies-arrayexpress

Deep sequencing data for PAM-SCANR tested CRISPR-Cas systems

Project description:In this work, we analyzed recognized PAM sequences from four CRISPR-Cas systems: E. coli I-E, B. halodurans I-C, S. thermophilus CR1 II-A, and F. novicida V. Cells containing functional PAMs were sorted using FACS and subsequently sequenced. Ranking of recognized PAMs from a positive screen

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Deep sequencing data for PAM-SCANR tested CRISPR-Cas systems

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A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

Project description:A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

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Expanding the Scope of Bacterial CRISPR Activation with PAM-flexible dCas9 Variants

Project description:CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective gene activation. While genes may not always have an NGG protospacer adjacent motif (PAM) at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the tradeoff that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPR interference (CRISPRi) gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems.

2022-11-15 | GSE217559 | GEO

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