Mallett Darren R, Chang Mingqin, Cheng Xiaohang, Bezanilla Magdalena
Department of Biological Sciences Dartmouth College Hanover New Hampshire.
Plant Biology Graduate Program University of Massachusetts Amherst Massachusetts.
Plant Direct. 2019 Sep 12;3(9):e00168. doi: 10.1002/pld3.168. eCollection 2019 Sep.
CRISPR-Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA-guided nuclease to initiate double-strand breaks. Until recently, classical RAD51-mediated homologous recombination has been a powerful tool for gene targeting in the moss . However, CRISPR-Cas9-mediated genome editing in was shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR-Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR-Cas9 genome editing in , here we describe the generation and use of a flexible and modular CRISPR-Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology-directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5' and 3' ends of gene coding regions. With regard to homology-directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a "stop codon cassette" with downstream near-effortless genotyping.
近年来，CRISPR - Cas9已被证明是一种有价值的工具，它使研究人员能够使用RNA引导的核酸酶精确编辑基因组，引发双链断裂。直到最近，经典的RAD51介导的同源重组一直是苔藓基因靶向的强大工具。然而，在[具体对象未提及]中，CRISPR - Cas9介导的基因组编辑被证明比传统同源重组更有效（《植物生物技术杂志》，2017年第15期，第122页）。CRISPR - Cas9提供了在多个位点高效编辑基因组的机会，以及使用简单的瞬时转化在基因组精确位置整合序列的机会。为了充分利用CRISPR - Cas9在[具体对象未提及]中的基因组编辑，在此我们描述一种灵活且模块化的CRISPR - Cas9载体系统的构建及使用。无需基因合成，该载体系统能够同时编辑多达12个位点。使用这个系统，我们生成了多个在四个不同位点具有无效等位基因的株系。我们还发现，在单个位点靶向多个位点可以产生更大的缺失，但这一成功取决于各个原间隔序列。为了利用同源定向修复，我们开发了模块化载体，以快速生成DNA供体质粒，从而在基因编码区的5'和3'端高效引入编码荧光蛋白的DNA序列。关于同源定向修复实验，我们发现，如果原间隔序列保留在DNA供体质粒上，那么Cas9会切割质粒靶点以及基因组靶点。这会降低将序列引入基因组的效率。此外，为了确保在Cas9切割位点附近产生无效等位基因，我们构建了一个携带“终止密码子盒”的同源质粒，并实现了近乎轻松的下游基因分型。
