Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum)
Application and optimization of CRISPR-Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum)
复制标题
CRISPR-Cas9介导的基因组工程在蝾螈（Ambystoma mexicanum）中的应用和优化
DOI：
10.1038/s41596-018-0071-0
复制
发表时间：
2018-12-01
期刊：
影响因子：
14.8
通讯作者：
Tanaka, Elly M.
中科院分区：
文献类型：
--
作者：
Fei, Ji-Feng;Lou, Wilson Pak-Kin;Tanaka, Elly M.
基因组操作是必不可少的模式生物的使用，以了解发展，再生和成年生理。蝾螈（Ambystoma mexicanum）是蝾螈的一种，在一系列复杂的组织和器官中表现出无与伦比的再生能力，因此可以作为解剖再生机制的强大动物模型。我们在这里描述了一个优化的逐步方案，使用CRISPR-Cas9系统创建遗传修饰的蝾螈。该方案需要7-8周才能完成，描述了靶向基因敲除和敲入的产生，并包括大靶向构建体的位点特异性整合。直接使用纯化的CAS9-NLS（含有C末端核定位信号的CAS9）蛋白允许引导RNA（gRNA）-CAS9-NLS核糖核蛋白（RNP）复合物的迅速形成，这加速了在单细胞期美西螈卵中靶向基因组基因座处的双链断裂（DSB）的产生。利用该方案，可以获得大量携带同源型移码突变的F-0个体，从而允许在这一代中进行表型分析。在靶向构建体的存在下，可以以非同源末端连接（NHEJ）方式以高达15%的效率将外源基因插入靶向美西螈基因组基因座中。我们的方案绕过了蝾螈的长世代时间，并允许在F-0基因操纵的蝾螈直接功能分析。该方案可以潜在地应用于其他动物模型，特别是具有良好表征的转录组但缺乏良好表征的基因组的生物体。
Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. The protocol, which takes 7-8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)-CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F-0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F-0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome.