I could not find a study that used CRISPR-mediated HDR for stable integration of large genes. I would be grateful if anyone could point me in the right direction.
Yes, CRISPR-mediated homology-directed repair (HDR) can be used for large gene production, including genes up to 4 kilobase pairs (kbp) in size. HDR is a mechanism that uses a donor template to precisely insert or modify DNA sequences at a specific genomic locus.
To produce large genes via CRISPR-mediated HDR, researchers typically design a gRNA to target the specific genomic site where they want to insert the large gene. They then co-transfect or co-inject the gRNA, Cas9 enzyme, and a donor template containing the desired large gene sequence into the target cells or embryos.
Efficient HDR can be more challenging with larger DNA fragments due to the increased likelihood of undesired outcomes, such as non-homologous end joining (NHEJ) repair or incomplete integration. Researchers often optimize experimental conditions and delivery methods to enhance HDR efficiency and minimize undesired outcomes.
Despite these challenges, significant progress has been made in improving HDR efficiency, making it a feasible approach for large gene production using CRISPR-Cas9 technology. As always, it is essential to stay updated with the latest methodologies and guidelines to achieve the best results in your experiments.
The latest technology now is Homology mediated end joining (HMEJ). It is more efficient than existing gene knock-in strategies in many systems (in vitro cultured cells, animal embryos, and in vivo tissues). Based on more efficient editing efficiency and better accuracy, HMEJ mediated gene tapping methods provide great application prospects for a series of applications, including the establishment of animal models for gene editing, targeted gene therapy for diseases, and so on.
Related references:
[1] Yao X, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res, 2017, 27(6): 801-14
[2] Xu J, et al. A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG. Stem Cell Res Ther, 2020, 11(1): 67
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