Mathew Saumya Mary
Central Animal Facility, Indian Institute of Science, Bangalore, 560 012, India.
Mol Biol Rep. 2023 Apr;50(4):3189-3204. doi: 10.1007/s11033-023-08278-8. Epub 2023 Jan 26.
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.
CRISPR/Cas9系统通常用于生成基因编辑小鼠模型。成簇规律间隔短回文重复序列基因编辑技术可利用非同源末端连接修复途径高效产生基因敲除。借助同源定向修复（HDR）机制，使用修复模板时小片段基因敲入也能精确实现。然而，当片段大小大于4-5 kb时，基因敲入往往容易出错且效率降低。某些类型的修饰，特别是插入非常大的DNA片段（10-100 kb）或整个基因替换，仍然具有挑战性。HDR过程需要进一步优化和改进。在本综述中，我们描述了从向导设计到显微注射以及卵母细胞供体选择的各个步骤来提高基因敲入效率的方法。这有助于理解哪些参数可以修改，以通过CRISPR靶向提高基因敲入效率。
