JGG:农科院水稻所王克剑研究组等创建简单高效的CRISPR-Cas9突变体
2017年4月4日，国际著名学术期刊《Journal of genetics and Genomics》杂志上在线发表了中国水稻研究所水稻染色体工程及基因组编辑创新团队王克剑研究组与苏州大学黄健课题组合作的一篇研究论文，研究基于常规聚合酶链式反应(PCR)原理，成功开发出临界退火温度PCR (ACT-PCR)用于快速高效地筛选基因编辑突变体。华宇峰、王春和苏州大学黄健为该论文的共同第一作者，王克剑研究员为通讯作者。
随着CRISPR-Cas9基因组编辑技术的广泛应用，科研人员需要进行大量的突变体筛选工作。目前通常的筛选方法包括对PCR产物限制性酶切、T7E1酶切、page分析、溶解曲线分析等，这些方法存在着耗时费钱、位点特异或者需要特定仪器等缺点。因此，迫切需要开发一种简单高效的筛选方法。
PCR是分子生物实验的一种常规技术，主要由变性—退火—延伸三个基本反应步骤构成。其中，特异性的引物以及适宜的退火温度是PCR反应能否成功扩增的关键因素。科研人员通过设计基因编辑位点特异引物，首先以野生型脱氧核糖核酸(DNA)为模板进行一次梯度PCR寻找出特异引物的临界退火温度，再在临界退火温度下进行PCR扩增筛选突变体。由于该特异引物不能和突变体DNA严格匹配，导致在临界退火温度下突变体中的PCR反应不能有效进行扩增。因此最终只需进行一次常规的PCR反应，便可以灵敏地检测出成功编辑的突变体。利用ACT-PCR，研究人员不仅在水稻中快速鉴定出单突变体和多突变体，同时也在斑马鱼中成功鉴定出突变体，表明ACT-PCR的应用不受物种的限制。除用于基因编辑突变体鉴定之外，研究还发现结合实时荧光定量pcr技术，ACT-PCR还可用于定量计算在细胞系中进行的基因编辑效率，与常用的PCR产物酶切等方法相比，通过定量ACT-PCR得到的数据更为接近于真实的编辑效率。
原文链接：
A simple and efficient method for CRISPR/Cas9-induced mutant screening
原文摘要：
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction (PCR)/restriction enzyme (RE) assay, T7 endonuclease I (T7EI) assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting (HRM) analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are time- and labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we describe a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR (ACT-PCR) for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.
作者：王克剑
