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

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

PROVIDER: PRJNA769264 | ENA |

REPOSITORIES: ENA

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

Project description: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.

2021-10-09 | GSE185486 | 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:A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

| PRJNA610555 | 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:Randomized PAM depletion of phylogenetically-diverse Cas12a nucleases

| PRJNA539942 | ENA

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

High targeted integration frequency in human pluripotent stem cells with the CRISPR-Cas12a Ultra system

Project description:The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypic Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated for the first time the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

2024-02-09 | GSE249731 | GEO

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

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

Project description: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.

2023-04-17 | GSE229888 | GEO

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.

2019-01-01 | GSE123443 | GEO

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