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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE249731 | GEO | 2024/02/09

REPOSITORIES: GEO

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Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption

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

Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing

Project description:Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows for robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.

2021-09-10 | GSE180773 | GEO

Project description:Allelic outcomes analysis after CRISPR-mediated editing

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Multiplex genome-editing in Arabidopsis thaliana using Mb3Cas12a

Project description:The use of CRISPR/Cas proteins for the creation of multiplex genome-engineering represents an important avenue for crop improvement, and further improvements for creation of knock-in plant lines via CRISPR-based technologies may enable the high-throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR/Cas9 system for multiplex genome-engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome-editing in Arabidopsis thaliana. We identified optimized promoter sequences for driving expression of single transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a-edited alleles at multiple target sites. By utilizing this system, we demonstrate single-transcript multiplexed genome-engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a-based genome-editing via whole-genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex-genome-engineering in plant-based systems.

2021-09-15 | GSE162875 | GEO

Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform

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

Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform

2020-02-29 | GSE144078 | GEO

Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

Project description:Genome editing typically involves recombination between donor nucleic acids and genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, amongst other deleterious by-products, DSBs yield translocations, off-target mutations and, most pervasively, unpredictable on-target allelic disruptions. Remarkably, hitherto, the untoward phenotypic consequences of on-target disruptions at allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness due to on-target DSBs and report that simultaneous single-stranded DNA break formation at donor and target DNA by CRISPR-Cas9 “nickases” overcomes, to a great extent, such genotype-phenotype disrupting events. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments (i.e. live-cell reporter tags) into essential and multiple-copy genomic sequences while circumventing most of the allelic and non-allelic collateral mutations and chromosomal rearrangements characteristic of nuclease-dependent gene editing procedures.

2019-12-04 | GSE135064 | GEO

A multi-functional AAV-CRISPR-Cas9 and its host response

Project description:CRISPR-Cas9 delivery by AAV holds promise for gene therapy but faces critical barriers due to its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multi-functional platform customizable for genome-editing, transcriptional regulation, and other previously impracticable AAV-CRISPR-Cas9 applications. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce detectable cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics mRNA-Seq from muscles (9 samples; 3 mice x 3 conditions) and lymph nodes (9 samples; 3 mice x 3 conditions).

2016-07-29 | E-GEOD-84534 | biostudies-arrayexpress

HBV-KMT2B integrated human induced pluripotent stem cells (KMT2B-Int iPSCs) vs heterozygous mutated KMT2B iPSCs (KMT2B-HT iPSCs) vs homozygous mutated KMT2B iPSCs (KMT2B-KO iPSCs) vs their original iPSCs (KMT2B-WT iPSCs)

Project description:Transcriptional profiling of all KMT2B-WT, HT, KO, Int iPSCs. KMT2B-HT, KO, and Int iPSCs were generated by gene editing using CRISPR/Cas9 and Cre recombinase technologies.

2023-11-15 | GSE217994 | GEO

Retroelement insertion in a CRISPR/Cas9 editing site in the early embryo intensifies genetic mosaicism

Project description:Continued CRISPR/Cas9-mediated editing activity that allows differential and asynchronous modification of alleles in successive cell generations expands allelic complexity. To understand the earliest events during CRISPR/Cas9 editing and the allelic selection among the progeny of subsequent cell divisions, we inspected in detail the genotypes of 4- and 8-cell embryos and embryonic stem cells (ESCs) after microinjection of a CRISPR toolkit into the zygotes. We found a higher editing frequency in 8-cell embryos than in 4-cell embryos, indicating that the CRISPR/Cas9 activity persisted through the 8-cell stage. Analysis of a CRISPR/Cas9 transgenic founder mouse revealed that four different alleles were present in its organs in different combinations and that its germline included three different mutant alleles, as shown by the genotypes of the pups. The indel depth, which measured the extent of indels at the sequence level within single embryos, decreased significantly as the embryos advanced to form ESCs, suggesting that exclusion of fatal indels occurred in the subsequent cell generations. Interestingly, we discovered that the CRISPR sites frequently contained introduced retroelement sequences and that this occurred preferentially with certain classes of retroelements. Therefore, in addition to CRISPR/Cas9’s innate mechanism of separate, differential enzymatic modifications of alleles, the frequent retroelement insertions that occur in early mouse embryos during CRISPR/Cas9 editing further expand the allelic diversity and mosaicism in the resulting transgenic founders.

2019-10-22 | GSE136393 | GEO

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