| IAA2 is a member of Aux/IAA auxin responsive gene family of Arabidopsi s thaliana. This gene family plays a very important role in plant growth and d evelopment, but there has not been reported IAA2 mutants, hindering the in-dep th study of its function and mechanism. Due to the gametophyte dominant, eas y to culture and prone to homologous recombination, Physcomitrella patens rep resents a new kind of model plant. The gene LrgB of Arabidopsis thaliana has been showed to associate with cell death regulation, Physcomitrella patens has two LrgB homologous genes, PpLrgBl and PpLrgB2, but their functions rema in unclear. CRISPR/Cas (clustered regulatory interspersed short palindromic rep eat- associated endonuclease) is an aquired immunity system in bacteria and ar chaea to resist exogenous virus or plasmid. Type Ⅱ CRISPR/Cas system has b een transformed into a highly efficient site-specific genome modification techno logy. Compared with other genome-editing techniques like the zinc finger endo nuclease (ZFN) and the transcription activation like effector nuclease (TALEN), CRISPR/Cas9 has several advantages such as simple operating process, high mutation efficiency and low cost. However, single gRNA only targets one geno me site, and the efficiency may not be high. Multiple gRNAs can target multi ple genome sites, thus, the efficiency of gene knock out may be improved. In the present paper, we developed a method to combine three gRNAs in the sa me entry vector, based on golden-gate cloning and two rounds of PCR. The fi nal expression vector was obtained by LR reaction with the destination vector containing the Cas9 expression cassette. Results showed that,4 of 6 gRNAs w ere effective, and a lot of insertion mutations and deletion mutations were obta ined. Compared with one gRNA approach, multiple gRNAs displayed higher ge ne knockout efficiency and produced more germ-line mutants. Compared with other methods to construct multiple gRNA vectors, our method was rapid and efficient. And we found that these mutants of NAA sensitivity is stronger, with longer petiole and root geotropism enhancement. Seven mutant lines generated here provided good materials for subsequent studies on IAA2 function. We als o got PpLrgBl and PpLrgB2 gene knockout clones of Physcomitrella patens, which require further identification.The following two questions were discussed in-depth:1. Two methods for construction of entry vector containing three gRNA expression cassettesThe two rounds of PCR method only uses 1 tube competent cells, and only needs one kind of type Ⅱs endonuclease; while the rounds of cloning method uses 4 tube competent cells and two kinds of type Ⅱs endonuclease. Two rounds of PCR method, therefore, save time and cost, seems more efficient.2. Comparison of two mutantion detection methodsCommon method for mutantion detection is T7 endonuclease enzyme digestion method, but its enzyme activity is susceptible to temperature, enzyme amount and the influence of reaction time, and sometimes there will generate false positive and false negative results. We used T7 endonuclease enzyme digestion method together with PAGE (polyacrylamide gel electrophoresis) method, and found that PAGE method has higher sensitivity and accuracy, low cost, and mutation types can be predicted according to the number and forms of band. But the process of making PAGE glue is relatively complex, in this case, agarose gel electrophoresis after T7 enzyme digestion seems more convenient. |
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