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ABSTRACT:
PROVIDER: PRJNA1168665 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing
| PRJNA610555 | ENA
Project description:Because of their smallness, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.
2022-09-12 | GSE213149 | GEO
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
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
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 | GSE145550 | GEO
Project description:we characterized a novel compact Cas12a ortholog, EbCas12a, from the Erysipelotrichia bacterium with activities in mammalian cells. It is with the PAM sequence of 5’-TTTV-3’ (V=A, G, C) and the smallest size of ~3.47kb among reported Cas12a orthologs so far. Moreover, enhanced EbCas12a (enEbCas12a) was also developed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells. With the help of the compact enEbCas12a, all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo. Altogether, with the help of the novel smallest high fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.
2023-07-10 | GSE236647 | GEO
Project description:Structures of RNA editing substrate binding complexes reveal the mechanisms of guide RNA stabilization and messenger RNA recognition in the Trypanosoma brucei mitochondrion.
2024-01-26 | PXD039156 | Pride
Project description:Systematic mapping of genetic interactions and interrogation of the functions of sizeable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR-based screening system for combinatorial genetic manipulation that employs co-expression of Cas9 and Cas12a nucleases and machine learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named CHyMErA (Cas Hybrid for Multiplexed Editing and Screening Applications), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive genetic interactions and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemo-genetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for genetic interaction mapping and the functional analysis of sizeable genomic regions, such as alternative exons.
2020-02-29 | GSE144078 | GEO
Project description:Systematic mapping of genetic interactions and interrogation of the functions of sizeable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR-based screening system for combinatorial genetic manipulation that employs co-expression of Cas9 and Cas12a nucleases and machine learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named CHyMErA (Cas Hybrid for Multiplexed Editing and Screening Applications), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive genetic interactions and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemo-genetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for genetic interaction mapping and the functional analysis of sizeable genomic regions, such as alternative exons.
2020-01-28 | GSE144281 | GEO