The typeⅡCRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) has been widely used in bacteria, yeast, animals and plants as a targeted genome editing tech-nique. In previous work, we have successfully knocked out the endogenous phytoene dehydrogenase (PDS) gene in Populus tomentosa Carr. using this system. To study the effect of target design on the efficiency of CRISPR/Cas9-mediated gene knockout in Populus, we analyzed the efficiency of mutagenesis using different sin-gle-guide RNA (sgRNA) that target PDS DNA sequence. We found that mismatches between the sgRNA and the tar-get DNA resulted in decreased efficiency of mutagenesis and even failed mutagenesis. Moreover, complementari-ty between the 3′ end nucleotide of sgRNA and target DNA is especially crucial for efficient mutagenesis. Further sequencing analysis showed that two PDS homologs in Populus, PtPDS1 and PtPDS2, could be knocked out simul-taneously using this system with 86.4% and 50% efficiency, respectively. These results indicated the possibility of introducing mutations in two or more endogenous genes efficiently and obtaining multi-mutant strains of Populus using this system. We have indeed generated several knockout mutants of transcription factors and structural genes in Populus, which establishes a foundation for future studies of gene function and genetic improvement of Populus.%CRISPR/Cas9系统是一种广泛应用于细菌、酵母、动物和植物中的基因组定点编辑技术。本课题组在前期工作中利用该系统在毛白杨(Populus tomentosa Carr.)中率先实现了对内源基因—八氢番茄红素脱氢酶(Phytoene dehydrogenase, PDS)基因的定点敲除。为研究靶点的设计和选择对该系统介导的杨树内源基因敲除效率的影响，本文分析了不同单向导RNA(Single-guide RNA, sgRNA)结合毛白杨PDS(PtPDS)靶基因DNA序列后对突变效率的影响。结果发现 sgRNA 与靶基因间的碱基错配会导致突变的效率降低，甚至不能突变，其中3′端的碱基配对更为重要。进一步测序分析发现，该系统能同时敲除杨树基因组上两个同源的 PDS 编码基因(PtPDS1和 PtPDS2)，突变率分别达86.4%和50%。研究证明该系统可快速高效地敲除两个以上的内源基因，获得多重突变体杨树株系。利用该技术，本课题组已获得多个杨树转录因子及结构基因的敲除突变体株系，为将来开展基因功能研究和杨树遗传改良奠定了基础。
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