The authors developed an optimized protocol for precise genome editing in primary human hematopoietic stem and progenitor cells (HSPCs) that can achieve near-perfect (>90%) editing efficiencies. Key elements of the protocol include:
The authors tested both adeno-associated virus (AAV) and short single-stranded oligodeoxynucleotide (ssODN) donors, and found that the ssODN donors could achieve similar near-perfect editing efficiencies when designed with the spacer-disrupting silent mutations.
Importantly, the editing was found to be even across the hematopoietic hierarchy, with no significant effects on progenitor phenotypes, lineage outputs, or the frequency of high self-renewal potential long-term culture-initiating cells.
The authors also demonstrated the ability to tune the zygosity of edited cells by providing a mixture of mutant and wild-type donor DNA. This enables the protocol to be useful for both therapeutic editing strategies and disease modeling applications.
Overall, this optimized protocol represents a significant advance in the field of precise genome editing in primary human HSPCs, opening new avenues for both curative treatments and accurate disease modeling.
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by Cloarec-Ung,... 場所 www.biorxiv.org 05-26-2023
https://www.biorxiv.org/content/10.1101/2023.05.26.542436v4深掘り質問