Details

Autor(en) / Beteiligte

• Mathew, Saumya Mary

Titel

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

Ist Teil von

• Molecular biology reports, 2023-04, Vol.50 (4), p.3189-3204

Ort / Verlag

Dordrecht: Springer Netherlands

Erscheinungsjahr

2023

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.