| Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences
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Author:
Date:
2017-09-20
[Abstract] Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products. Targeting of Cas9 to multiple genomic positions (termed ‘multiplexing’) is achieved by the expression of multiple sgRNAs within the same nucleus. However, an ongoing concern of the CRISPR field has been the accidental targeting of Cas9 to alternative (‘off-target’) DNA locations within a genome. We ...
[摘要] 鉴于CRISPR（集群定期间隔短回归重复）编辑技术的出现，基因组操纵变得更加易于使用。 Cas9核酸内切酶将募集复合物的单链（单向导）RNA（sgRNA）片段结合到相应的基因组靶序列，引发双链断裂。真核修复系统允许引入外源DNA，修复现有突变或内源基因产物的缺失。通过在同一核内表达多个sgRNA来实现Cas9对多个基因组位置的定位（称为“多重”）。然而，CRISPR领域的持续关注是将Cas9意外地定位到基因组内的替代（“脱靶”）DNA位置。我们将安装的人造Cas9靶序列的使用（称为人造基因座上的Cas9复制）描述为允许（i）与单个sgRNA复用的酵母基因组中的用途; （ii）减少/消除可能的脱靶效应，以及（iii）精确控制预定目标序列的放置。
【背景】CRISPR（集群定期间隔回归重复）机制已经在原核生物中演变为具有很高精度编辑任何基因组的能力的原始适应性免疫系统（Jinek等，2012; Sorek等，2013）。这种生物技术需要使用来自化脓性链球菌（或othologous物种）的内切核酸酶（Cas9），单个RNA'引导'序列和外源供体DNA（如果需要）。仅在短短几年内，CRISPR / ...
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| Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome
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Author:
Date:
2016-11-20
[Abstract] Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives.
[摘要] 由于其大尺寸，细菌致病性岛和其它连续操纵子可能难以使用常规方法克隆。在这里我们描述了一个强大的3步法从毒力质粒或粘粒转移大型定义的片段到更小的自主复制质粒或直接到细胞染色体，并入内源性酵母和λ红色同源重组系统的定义网站。该方法已经成功地用于分离和整合至少31kb的连续DNA，并且可以容易地适应于E的重组。大肠杆菌及其近亲。
[背景] 分离和繁殖大片DNA的能力大大扩展了基因网络和操纵子的研究。然而，用于该目的的传统使用的工程质粒，例如细菌人工染色体（BAC），虽然极其有用，但是受到DNA稳定性，拷贝数和复杂装配要求的问题的限制。或者，将构建体直接并入细菌染色体中通过减少由于存在多个基因拷贝引起的基因表达的变化以及确保基因的稳定维持而提供了优点，同时还避免了对抗生素选择的需要。这里描述的方法最初被设计为捕获和转移编码Shigella flexneri 3型分泌系统的31kb DNA操纵子到大肠杆菌染色体上（Reeves ...
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| Cell-based Assays to Monitor AID Activity
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Author:
Date:
2016-02-05
[Abstract] The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which provides an indication of its catalytic activity; 2) the capacity of AID to perform SHM by complementing a derivative of the DT40 chicken B cell line; 3) the ability of AID to perform CSR by complementing ...
[摘要] 酶活化诱导的脱氨酶（AID）通过其将脱氧胞苷脱氨基到DNA中的脱氧尿苷的诱变活性来支持抗体亲和力成熟和同种型转换。脱氧尿苷的后续加工引发B细胞中体细胞超突变（SHM）和类型转换重组（CSR）的过程。 AID的结构功能分析需要灵敏和生物相关的方法来测量其各种活动。在这里我们描述简单但有效的方法来测量1）AID突变大肠杆菌基因组的能力，其提供其催化活性的指示; 2）AID通过补充DT40鸡B细胞系的衍生物来进行SHM的能力; 3）AID通过补充AID缺乏的原代小鼠B细胞来进行CSR的能力。三种方法的组合，伴随着AID亚细胞定位和蛋白质表达水平和稳定性的必要分析作为对照，允许AID的详细结构功能研究。
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