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Error-free recombination in sugarcane mediated by only 30 nucleotides of homology and CRISPR/Cas9 induced DNA breaks or Cre-recombinase

机译:甘蔗中的无差异重组仅由同源性和CRISPR / CAS9诱导的DNA断裂或Cre-Refombinase的仅30个核苷酸介导

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Precision genome editing by homology directed repair has tremendous potential for crop improvement. This study describes in planta homologous recombination mediated by CRISPR/Cas9 induced DNA double strand break in proximity to a single short (similar to 30 nt) homology arm. The efficiency of CRISPR/Cas9-mediated recombination between two loxP sites was compared with Cre (Cyclization recombination enzyme) and codon-optimized Cre-mediated site-specific recombination in sugarcane. A transgenic locus was generated with a selectable nptII coding sequence with terminator between two loxP sites located downstream of a constitutive promoter and acting as transcription block for the downstream promoter-less gusA coding sequence with terminator. Recombination between the two loxP sites resulted in deletion of the transcription block and restored gus activity. This transgenic locus provided an efficient screen for identification of recombination events in sugarcane callus following biolistic delivery of Cre, codon-optimized Cre, or the combination of sgRNA and Cas9 targeting the 5 ' loxP site. The Cre codon optimized for sugarcane displayed the highest efficiency in mediating the recombination that restored gus activity followed by cre and CRISPR/Cas9. Remarkably the short region of homology of the loxP site cleaved by Cas9 (30 nt)-mediated error-free recombination in all 21 events from three different experiments that were analyzed by Sanger sequencing consistent with homology directed repair. These findings will inform rational design of strategies for precision genome editing in plants.
机译:通过同源性定向修复的精确基因组编辑具有巨大的作物改善潜力。本研究描述了由CRISPR / CAS9诱导的DNA双链介导的植物同源重组,邻近单个短(类似于30nt)同源臂。将2个LOXP位点之间的CRISPR / CAS9介导的重组的效率与CRE(环化重组酶)和密码酮优化的CRE介导的位点特异性重组进行比较。通过选择性NPTII编码序列产生转基因座,其具有位于组成型启动子下游的两个LOXP位点之间的终止子,并用终止剂作用为下游启动子的GUSA编码序列的转录嵌段。两种LOXP位点之间的重组导致缺失转录块并恢复GUS活动。该转基因基因座提供了一种有效的筛选,用于鉴定CRE,密码子优化CRE或SGRNA和CAS9的组合靶向CRE,密码子优化CRE或靶向5'OXP位点的CARN9的组合后甘蔗愈伤组织中的重组事件。优化为甘蔗优化的CreCodon在介导恢复GUS活动之后的重组后介绍了最高效率,然后是CRE和CRISPR / CAS9。显着通过Cas9(30nt)介导的LOXP位点的性状的短区域 - 来自三种不同实验的所有21例事件中的Cas9(30nt)介导的无差异重组,所述三种不同实验中通过与同源性定向修复一致的Sanger测序分析。这些调查结果将向植物中的精确基因组编辑进行理性设计。

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