Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2023 Apr;50(4):3189-3204.
doi: 10.1007/s11033-023-08278-8. Epub 2023 Jan 26.

Strategies for generation of mice via CRISPR/HDR-mediated knock-in

Saumya Mary Mathew  1
Affiliations
Review

Strategies for generation of mice via CRISPR/HDR-mediated knock-in

Saumya Mary Mathew. Mol Biol Rep. 2023 Apr.

Abstract

CRISPR/Cas9 framework is generally used to generate genetically modified mouse models. The clustered regularly interspaced short palindromic repeat gene editing technique, can efficiently generate knock-outs using the non-homologous end-joining repair pathway. Small knock-ins also work precisely using a repair template with help of homology-directed-repair (HDR) mechanism. However, when the fragment size is larger than 4-5 kb, the knock-in tends to be error prone and the efficiency decreases. Certain types of modifications, in particular insertions of very large DNA fragments (10-100 kb) or entire gene replacements, are still difficult. The HDR process needs further streamlining and improvement. Here in this review, we describe methods to enhance the efficiency of the knock-in through checking each step from the guide design to the microinjection and choice of the oocyte donors. This helps in understanding the parameters that can be modified to get improved knock-in efficiency via CRISPR targeting.

Keywords: CRISPR; HDR; Knock-in efficiency; Microinjection; Targeted knock-ins.

PubMed Disclaimer

References

    1. Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6(6):507. https://doi.org/10.1038/nrg1619 - DOI - PubMed
    1. Cui X, Ji D, Fisher DA, Wu Y, Briner DM, Weinstein EJ (2011) Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol 29(1):64–67. https://doi.org/10.1038/nbt.1731 - DOI - PubMed
    1. Ding Q et al (2013) A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell 12(2):238–251. https://doi.org/10.1016/j.stem.2012.11.011 - DOI - PubMed
    1. Wang H et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/cas-mediated genome engineering. Cell 153(4):910–918. https://doi.org/10.1016/j.cell.2013.04.025 - DOI - PubMed - PMC
    1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (1979) 337(6096):816. https://doi.org/10.1126/science.1225829 - DOI

LinkOut - more resources