Hello.
I intend to insert a 20kb gene cassette into human genome using the CRISPR-Cas9 approach coupled with homology arm directed repair. It is know that this approach is less efficient due to the rare event of homology arm repairs, but it yields specific insertion at targeted loci. With such large construct, the probability in obtaining a clone will be extremely challenging.
I am seeking for advice and suggestions from anyone who has experience performing knock-in with such large construct, and which strategy was used specifically on human embryonic stem cell line.
Thank you.
We are also preparing to do this. We have recently reviewed some literature and hope it can help you.
[1] 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
[2] Zhi M, et al. Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Cell Research, 2022, 32(4): 383-400
[3] Ran FA, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 2013, 8(11): 2281-308
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