| DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis
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Author:
Date:
2017-06-05
[Abstract] We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set of tools, named CRISPR RGEN tools (http://www.rgenome.net/), which provides all required tools from CRISPR target design to NGS data analysis.
[摘要] 我们通过使用无DNA的CRISPR成功地引入了目标克隆在赖氨酸衣藻中的目标基因敲除。在该协议中，整个工作流程的详细过程涵盖从初始目标选择CRISPR到使用下一代测序（NGS）技术的突变体分析。此外，我们介绍一种基于Web的工具集，名为CRISPR RGEN工具（ http：// www.rgenome.net/ ），其中提供了CRISPR目标设计到NGS数据分析的所有必需工具。
背景 我们最近报道（Baek等人，2016），使用预先组装的Cas9蛋白质指导RNA核糖核蛋白，模型绿色微藻（Chlamydomonas ...
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| Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
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Author:
Date:
2017-05-20
[Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for ...
[摘要] 为了实现克氏锥虫内源蛋白的C末端标记，我们使用Cas9 / pTREX-n载体（Lander等，2015）在3'末端插入特异性标签序列（3xHA或3xc-Myc）特定的感兴趣基因（GOI）。将靶向GOI 3'末端的嵌合sgRNA进行PCR扩增，并克隆到Cas9 / pTREX-n载体中。然后扩增通过同源重组诱导DNA修复的DNA供体分子。该供体序列包含标签序列和抗生素抗性标记，加上对应于位于终止密码子右上游的区域和在GOI基因座的Cas9靶位点下游的100bp同源臂。载体pMOTag23M（Oberholzer等，2006）或pMOHX1Tag4H（Lander等，2016b）用作DNA供体扩增的PCR模板。用sgRNA / Cas9 / pTREX-n构建体和DNA供体盒共转染的Epimastigotes然后用抗生素培养5周以选择双重抗性寄生虫。最终通过PCR和Western印迹分析验证了内源基因标记。
【背景】自从CRISPR / ...
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