Dataset Information

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

ABSTRACT: The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical 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.

SUBMITTER: Hammad R

PROVIDER: S-EPMC10816062 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Publications

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

Hammad Ruba R Alzubi Jamal J Rhiel Manuel M Chmielewski Kay O KO Mosti Laura L Rositzka Julia J Heugel Marcel M Lawrenz Jan J Pennucci Valentina V Gläser Birgitta B Fischer Judith J Schambach Axel A Moritz Thomas T Lachmann Nico N Cornu Tatjana I TI Mussolino Claudio C Schäfer Richard R Cathomen Toni T

International journal of molecular sciences 20240112 2

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical 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 enab ...[more]

PMID: 38256061

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Project description:Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects. CRISPR delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects, and reduce cellular toxicity. Although RNP delivered CRISPR genome editing has been demonstrated in many plant species, optimization strategies that yield high editing efficiencies have not been thoroughly investigated. Using rice and citrus protoplast systems we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a, and AsCas12a Ultra, were investigated. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L, and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP) and verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32°C and 25°C. LbCas12a and LbCas12a-E795L demonstrated increased editing efficiencies in comparison to AsCas12a and AsCas12a Ultra, especially when used at lower RNP concentrations. In addition, we discovered that a 1:1 Cas12a:crRNA molar ratio is sufficient to achieve efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP-based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained similarly high editing efficiencies at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants.

Dataset Information

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

Publications

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

Similar Datasets

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