| Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap
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Author:
Date:
2018-10-20
[Abstract] This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any endogenous full-length transcript from the target gene. CRISPR-Trap is applicable for any target gene with no or little coding sequence in its first exon. Several human cell lines and different genes have so far been edited successfully with CRISPR-Trap.
[摘要] 该协议描述了CRISPR-Trap从设计基因靶向策略到验证成功编辑的克隆的应用，所述克隆在各种人细胞系上得到验证，其中人类诱导的多能干细胞（hiPSC）。 CRISPR-Trap优于常规方法的优点是从靶基因完全去除任何内源全长转录物。 CRISPR-Trap适用于在其第一个外显子中没有编码序列或编码序列很少的任何靶基因。 到目前为止，已经使用CRISPR-Trap成功编辑了几种人细胞系和不同基因。
【背景】CRISPR / Cas9技术的出现促进了基因敲除和基因编辑的基因组靶向。执行敲除的常规方法依赖于引入移码导致过早终止密码子（PTC），截短开放阅读框（ORF）以及随后通过无义介导的mRNA衰变（NMD）降解靶基因的转录物。 。这种方法的一个可能的缺陷是全长转录物，其可以逃避NMD并产生具有残余或甚至显性负功能的C末端截短蛋白。该协议提出了CRISPR-Trap，这是我们最近建立的一种方法（Reber et al。>，2018），成功编辑后将阻止从靶基因位点表达任何全长转录本（图1）。简而言之，这种方法针对CRISPR / ...
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| RNA Immunoprecipitation (RIP) Sequencing of Pri-miRNAs Associated with the Dicing Complex in Arabidopsis
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Author:
Date:
2018-07-05
[Abstract] RNA immunoprecipitation (RIP) is an antibody-based technique used to map in vivo RNA-protein interactions. DBR1, an RNA debranching enzyme, is responsible for the debranching of lariat RNA, for the degradation and turnover of lariat RNAs. It is well known that primary miRNA (Pri-miRNA) is recognized and further processed into mature miRNA by the Dicing complex mainly composed of DCL1 and HYL1. Due to the low abundance of pri-miRNAs, RIP followed qRT-PCR has been widely used to evaluate the binding efficiency of the Dicing complex with pri-miRNAs in previous studies. Therefore, the genome-wide evaluation of the Dicing complex with pri-miRNAs is lacking. With the improvement of high-throughput sequencing technologies, we successfully used RIP-seq to compare the binding efficiency ...
[摘要] RNA免疫沉淀（RIP）是一种基于抗体的技术，用于绘制体内 RNA-蛋白质相互作用。 DBR1是一种RNA脱支酶，负责套索RNA的脱支，用于套索RNA的降解和转换。众所周知，主要miRNA（Pri-miRNA）被主要由DCL1和HYL1组成的切割复合物识别并进一步加工成成熟miRNA。由于pri-miRNA的丰度较低，RIP随后的qRT-PCR已被广泛用于评估切割复合物与pri-miRNA在先前研究中的结合效率。因此，缺乏对具有pri-miRNA的切割复合物的全基因组评估。随着高通量测序技术的改进，我们成功地使用RIP-seq比较了Dicing复合物与野生型和 dbr1-2 突变体之间的pri-miRNA的结合效率。 。在该方案中，我们提供了在两种不同基因型之间的HYL1-YFP和DCL1-YFP转基因植物中使用GFP捕获珠的RIP-seq的详细描述。该方法可用于评估pri-miRNA与拟南芥中的切割复合物的结合，并且它可以应用于植物中的其他RNA结合蛋白。
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| Circular RT-PCR Assay Using Arabidopsis Samples
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Author:
Date:
2015-07-20
[Abstract] Post-transcriptional processing is critical for RNA biogenesis, in which conventional functional RNA transcripts are generated, such as messenger RNAs (mRNAs), transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) for translation as well as emerging non-coding RNAs with known or unknown regulatory functions. To determine the precise termini of an RNA molecule during or after processing, the primer extension and Rapid Amplification of cDNA Ends (RACE) methods have been routinely utilized for the precise mapping of 5’ or 3’ ends. Different from these assays, which are designed to detect only one end of a specific target RNA at a time, circular Reverse Transcription-Polymerase Chain Reaction (cRT-PCR) is able to simultaneously determine both the 5’ and 3’ ends of the target RNA. In Arabidopsis ...
[摘要] 转录后加工对于RNA生物发生至关重要，其中产生常规功能性RNA转录物，例如用于翻译的信使RNA（mRNA），转移RNA（tRNA）和核糖体RNA（rRNA）以及已知的新编码RNA或未知的调节功能。为了在加工期间或之后确定RNA分子的精确末端，引物延伸和cDNA末端的快速扩增（RACE）方法已经常规地用于5'或3'末端的精确作图。与设计为一次仅检测特定靶RNA的一端的这些测定法不同，环状逆转录 - 聚合酶链式反应（cRT-PCR）能够同时测定靶标的5'和3'末端RNA。在拟南芥中，cRT-PCR已经被广泛应用于鉴定核糖体RNA前体的5'和3'末端，或者评估在3'末端的长度或转录后延伸成熟mRNA。在该方案中，我们总结并提出了在拟南芥中cRT-PCR测定的详细程序，其也成功地用于我们以前公开的工作中（Hang等人 ，2014）。
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