Abstract：
BACKGROUND: In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated.
RESULTS: We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions.
CONCLUSIONS: Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.
RESULTS: We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions.
CONCLUSIONS: Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.
摘要：
背景：在植物基因组编辑中，RNA引导的核酸酶如来自化脓性链球菌的Cas9(SpCas9)主要在靶位点处诱导小的插入或缺失。这可用于通过移码突变使蛋白质编码基因失活。然而,在某些情况下,删除较大的染色体片段可能是有利的。这通过同时诱导待缺失区段的上游和下游的双链断裂来实现。尚未系统地评估删除较大染色体片段的实验方法。
结果：我们设计了三对指导RNA，用于缺失包含拟南芥WRKY30基因座的〜2.2kb染色体片段。我们测试了指导RNA对的组合和外切核酸酶TREX2的共表达如何影响编辑实验中wrky30缺失的频率。我们的数据表明，与一对引导RNA相比，两对增加染色体缺失的频率。外切核酸酶TREX2增强了个体靶位点处的突变频率,并使突变谱向更大的缺失转移。然而,TREX2没有增加染色体片段缺失的频率。
结论：使用至少两对指导RNA（总共四个指导RNA）的多重编辑至少在AtWRKY30基因座上提高了染色体片段缺失的频率，从而简化了相应突变体的选择。TREX2外切核酸酶的共表达可以用作提高拟南芥编辑效率的一般策略，而没有明显的负面影响。
结果：我们设计了三对指导RNA，用于缺失包含拟南芥WRKY30基因座的〜2.2kb染色体片段。我们测试了指导RNA对的组合和外切核酸酶TREX2的共表达如何影响编辑实验中wrky30缺失的频率。我们的数据表明，与一对引导RNA相比，两对增加染色体缺失的频率。外切核酸酶TREX2增强了个体靶位点处的突变频率,并使突变谱向更大的缺失转移。然而,TREX2没有增加染色体片段缺失的频率。
结论：使用至少两对指导RNA（总共四个指导RNA）的多重编辑至少在AtWRKY30基因座上提高了染色体片段缺失的频率，从而简化了相应突变体的选择。TREX2外切核酸酶的共表达可以用作提高拟南芥编辑效率的一般策略，而没有明显的负面影响。
