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Efficient Gene-Editing in Human Pluripotent Stem Cells Through Simplified Assembly of Adeno-Associated Viral (AAV) Donor Templates
- Source :
- Bio-Protocol, Vol 14, Iss 21 (2024)
- Publication Year :
- 2024
- Publisher :
- Bio-protocol LLC, 2024.
-
Abstract
- Gene-edited human pluripotent stem cells provide attractive model systems to functionally interrogate the role of specific genetic variants in relevant cell types. However, the need to isolate and screen edited clones often remains a bottleneck, in particular when recombination rates are sub-optimal. Here, we present a protocol for flexible gene editing combining Cas9 ribonucleoprotein with donor templates delivered by adeno-associated virus (AAV) vectors to yield high rates of homologous recombination. To streamline the workflow, we designed a modular system for one-step assembly of targeting vectors based on Golden Gate cloning and developed a rapid protocol for small-scale isolation of AAV virions of serotype DJ. High homology-directed repair (HDR) rates in human pluripotent stem cells (hPSCs), ~70% in ACTB and ~30% in LMNB1, were achieved using this approach, also with short (300 bp) homology arms. The modular design of donor templates is flexible and allows for the generation of conditional and/or complex alleles. This protocol thus provides a flexible and efficient strategy workflow to rapidly generate gene-edited hPSC lines.
- Subjects :
- Biology (General)
QH301-705.5
Subjects
Details
- Language :
- English
- ISSN :
- 23318325
- Volume :
- 14
- Issue :
- 21
- Database :
- Directory of Open Access Journals
- Journal :
- Bio-Protocol
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.32857062d4764758a4c154ecca61ce0a
- Document Type :
- article
- Full Text :
- https://doi.org/10.21769/BioProtoc.5097