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
- Location
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Deutsche Nationalbibliothek Frankfurt am Main
- Extent
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Online-Ressource
- Language
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Englisch
- Notes
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International journal of molecular sciences. - 25, 2 (2024) , 985, ISSN: 1422-0067
- Event
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Veröffentlichung
- (where)
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Freiburg
- (who)
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Universität
- (when)
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2024
- Creator
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Hammad, Ruba
Alzuʾbi, Jamal
Rhiel, Manuel
Chmielewski, Kay O.
Mosti, Laura
Rositzka, Julia
Heugel, Marcel
Lawrenz, Jan
Pennucci, Valentina
Gläser, Birgitta
Fischer, Judith
Schambach, Axel
Moritz, Thomas
Lachmann, Nico
Cornu, Tatjana I.
Mussolino, Claudio
Schäfer, Richard
Cathomen, Anton
- DOI
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10.3390/ijms25020985
- URN
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urn:nbn:de:bsz:25-freidok-2435574
- Rights
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Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
- Last update
- 15.08.2025, 7:24 AM CEST
Data provider
Deutsche Nationalbibliothek. If you have any questions about the object, please contact the data provider.
Associated
- Hammad, Ruba
- Alzuʾbi, Jamal
- Rhiel, Manuel
- Chmielewski, Kay O.
- Mosti, Laura
- Rositzka, Julia
- Heugel, Marcel
- Lawrenz, Jan
- Pennucci, Valentina
- Gläser, Birgitta
- Fischer, Judith
- Schambach, Axel
- Moritz, Thomas
- Lachmann, Nico
- Cornu, Tatjana I.
- Mussolino, Claudio
- Schäfer, Richard
- Cathomen, Anton
- Universität
Time of origin
- 2024
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