通过CRISPR-Cas9介导建立可诱导的条件性敲除小鼠模型

CRISPR/Cas9 technology is a versatile tool for genome engineering, developed from a adaptive immune defenses of bacteria and archaea in 2013. It use short guide RNA to recognize and direct degradation of targeted DNA by Cas9 nucleases, triggering non-homologous end joining (NHEJ)- mediated disruption of genome or homologous recombination (HR) –mediated modification of the genome. Compared with other gene editing technologies, CRISPR/Cas9 is scalable, affordable and easy to engineer and achieved genomic multiple–site modifications. Based on these characteristics, we aim to generate inducible and conditional knockout mouse model by modifying and optimizing Cas9 system. Site specific integration of Cas9 and U6-sgRNA into a tissue-specific gene would cause a tissue-specific Cas9 expression. And meanwhile, we fuse bacterial dihtdrofolate reductase (ecDHFR) to Cas9 nucleases. Cas9 nucleases would be degraded after translation due to following the ecDHFR structure, but the decay can be blocked by injection of trimethoprim (TMP). Thus, after integrating ecDHFR-Cas9-U6-sgRNA structure into a tissue-specific gene, tissue-specific knockout mice would be generated with TMP induction.

CRISPR-Cas9技术是2013年初由细菌和古细菌中的CRISPR免疫防御系统改造建立的一种基因组编辑新工具。它利用一段可以识别特定DNA序列的短RNA序列引导，由Cas9对靶向序列进行切割，从而引发同源重组或非同源末端连接导致的序列突变。CRISPR/Cas9技术相比其它的基因组修饰技术，具有容易构建、容易规模化、多靶向修饰的特点。 由此，我们拟对Cas9进行改造和优化，从而建立Cas9介导的可诱导的条件性敲除小鼠模型：将Cas9和U6-sgRNA元件定点整合到某一特定基因的后面，使Cas9与某一特定基因同时表达，从而Cas9发生组织特异性的表达。同时将Cas9与ecDHFR融合，可以使Cas9在翻译后迅速降解，只有在注射甲胺苄腚的情况下，Cas9蛋白才能稳定不被降解。因此，将ecDHFR-Cas9-U6-sgRNA整合到小鼠某一特定基因后，可以在甲胺苄腚的诱导下得到条件敲除小鼠。

利用Cas9介导对MECP2特定基因敲入甲胺苄啶诱导调控的小鼠模型构建成功，但是甲胺苄腚的诱导下并没有得到条件敲除小鼠，提示我们构建的载体在体内可能失去了预期的功能作用，提示了体内特定基因调控的复杂性。.在此基础上，我们对小鼠模型体内体外的筛选体系，以及特定模型建立和小鼠早期胚胎发育做出了成绩：建立了以小鼠单倍体干细胞为基础的单拷贝的筛选系统，证实Cyclin B3蛋白引起小鼠胚胎早期致死，证明了小鼠4细胞，8细胞单卵裂球的全能性，这些工作对于基于小鼠为研究对象的基因编辑模型的建立具有理论和技术支撑作用。