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

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

PROVIDER: PRJNA610555 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11244000.fastq.gz	Fastqsanger.gz
	SRR11244100_1.fastq.gz	Fastqsanger.gz
	SRR11244100_2.fastq.gz	Fastqsanger.gz
	SRR11244001.fastq.gz	Fastqsanger.gz
	SRR11244101.fastq.gz	Fastqsanger.gz

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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

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

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

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

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Cas12a base editors induce efficient and specific editing with low DNA damage response (RNA sequencing)

Project description:The advent of base editors (BEs) holds a promising potential in correcting pathogenic-related point mutations to treat relevant diseases. Unexpectedly, Cas9 nickase (nCas9) derived BEs lead to DNA double-strand breaks, which can trigger unwanted cellular responses including a p53-mediated DNA damage response (DDR). Here, we showed that catalytically-dead-Cas12a (dCas12a) conjugated BEs induced no DNA break and minimally activated DDR proteins including H2AX, ATM, ATR and p53. We further developed a BEACON (Base Editing induced by human APOBEC3A and Cas12a without DNA break) system that fuses dCas12a to the engineered APOBEC3A with enhanced deamination efficiency and editing specificity. By using BEACON, efficient C-to-T editing was achieved at levels comparable to AncBE4max and only low levels of DDR and RNA off-target (OT) effects were triggered in mammalian cells. BEACON also induced in vivo base editing in mouse embryos and targeted C-to-T conversions were detected in F0 mice.

2020-05-05 | GSE144278 | GEO

Cas12a base editors induce efficient and specific editing with low DNA damage response (DNA targeted sequencing)

2020-05-05 | GSE145550 | GEO

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

A Cas12a ortholog with distinct TTNA PAM enables sensitive detection of HPV16/18.

Project description:CRISPR-associated (Cas) nucleases are multifunctional tools for gene editing. Cas12a possesses several advantages, including the requirement of a single guide RNA and high fidelity of gene editing. Here, we tested three Cas12a orthologs from human gut samples and identified a LtCas12a that utilizes a TTNA protospacer adjacent motif (PAM) distinct from the canonical TTTV PAM but with equivalent cleavage ability and specificity. These features significantly broadened the targeting scope of Cas12a family. Furthermore, we developed a sensitive, accurate, and rapid human papillomavirus (HPV) 16/18 gene detection platform based on LtCas12a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) and lateral flow assay (LFA). LtCas12a showed comparable sensitivity to quantitative polymerase chain reaction (qPCR) and no cross-reaction with 13 other high-risk HPV genotypes in detecting the HPV16/18 L1 gene. Taken together, LtCas12a can broaden the applications of the CRISPR-Cas12a family and serve as a promising next-generation tool for therapeutic application and molecular diagnosis.

| S-EPMC10162949 | biostudies-literature

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

Project description:Cas12a is an RNA-guided endonuclease that has been widely used for convenient multiplex gene editing with low off-target effects. To minimize off-targeting in gene editing, we engineered a variant of LbCas12a (termed Lb-K538R) with more stringent PAM recognition, lower off-targeting capability, and similar editing efficiency in vivo compared with LbCas12a. We also demonstrated that Lb2Cas12a from Lachnospiraceae bacterium MA2020 has extensive gene-editing activities in mammalian cells. Similar to Lb-K538R, the designed Lb2Cas12a variant (termed Lb2-K518R) not only had a more stringent PAM sequence change from YYN to TYN (Y is T or C, N is A, T, C, or G), but also displayed lower off-target effects, thereby enabling more potential target site selections with low off-targeting than the common TTTV (V is A, G, or C) PAM. To determine whether this type of mutation at the homologous position had similar effects in other Cas12a, As-K548R was evaluated. Based on the results of the genome-wide off-target test, As-K548R displayed lower off-target effects. Collectively, our findings indicate that the Cas proteins could be designed to be stringent in PAM recognition to reduce their off-target effects, which suggests a promising and practical approach for minimizing off-targets effects in genome editing.

| S-EPMC8753454 | biostudies-literature

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