Highly Selective, CRISPR/Cas9-Mediated Isolation of Genes and Genomic Loci from Complex Genomes by TAR Cloning in Yeast.
Highly Selective, CRISPR/Cas9-Mediated Isolation of Genes and Genomic Loci from Complex Genomes by TAR Cloning in Yeast.
复制标题
DOI：
10.1002/cpz1.207
复制
发表时间：
2021-08
期刊：
影响因子：
--
通讯作者：
Larionov V
中科院分区：
文献类型：
--
作者：
Kouprina N;Kim JH;Larionov V
在这里，我们描述了一个更新的TAR克隆协议的选择性和有效的分离任何基因组片段或基因的利益，从基因组DNA的大小高达280 kb。该方法利用了酿酒酵母的特殊重组机制。TAR克隆基于目标特定基因组DNA片段与TAR载体中与靶向区域5′和3′端同源的靶向序列（钩）之间发生的高水平体内重组。在共转化到酵母中时，这导致感兴趣的染色体区域作为环状YAC分子分离，然后其在酵母细胞中繁殖和分离，并且可以被选择。在本文描述的更新的TAR克隆方案中，通过用专门设计的CRISPR/Cas9内切核酸酶预处理基因组DNA，通常获得的区域阳性克隆的分数从1%增加到35%，所述CRISPR/Cas9内切核酸酶产生包围靶基因组DNA序列的双链断裂（DSB），从而使感兴趣的染色体区域的末端高度重组。此外，还构建了一种新的TAR载体，其中包含YAC和BAC盒，允许将TAR克隆的DNA从酵母直接转移到细菌细胞。一旦构建了带有钩子的TAR载体并制备了基因组DNA，整个过程需要3周才能完成。更新后的TAR方案不需要大量的酵母经验或大量耗时的酵母工作，因为仅筛选约12个酵母转化体通常足以找到具有感兴趣区域的克隆。染色体片段、单个基因或基因家族的TAR克隆可用于功能、结构和群体研究，用于比较基因组学和长距离单体型分析，并具有基因治疗的潜力。2021年出版。本条目属于美国政府作品，在美国属于公有领域。由Wiley Periodicals LLC出版。 基本方案1：基础方案3：将TAR/YAC/BAC分离物从酵母转移至大肠杆菌（E.杆菌
Here we describe an updated TAR cloning protocol for the selective and efficient isolation of any genomic fragment or gene of interest up to 280 kb in size from genomic DNA. The method exploits the special recombination machinery of the yeast Saccharomyces cerevisiae. TAR cloning is based on the high level of in vivo recombination that occurs between a specific genomic DNA fragment of interest and targeting sequences (hooks) in a TAR vector that are homologous to the 5′ and 3′ ends of the targeted region. Upon co‐transformation into yeast, this results in the isolation of the chromosomal region of interest as a circular YAC molecule, which then propagates and segregates in yeast cells and can be selected for. In the updated TAR cloning protocol described here, the fraction of region‐positive clones typically obtained is increased from 1% up to 35% by pre‐treatment of the genomic DNA with specifically designed CRISPR/Cas9 endonucleases that create double‐strand breaks (DSBs) bracketing the target genomic DNA sequence, thereby making the ends of the chromosomal region of interest highly recombinogenic. In addition, a new TAR vector was constructed that contains YAC and BAC cassettes, permitting direct transfer of a TAR‐cloned DNA from yeast to bacterial cells. Once the TAR vector with the hooks is constructed and genomic DNA is prepared, the entire procedure takes 3 weeks to complete. The updated TAR protocol does not require significant yeast experience or extensively time‐consuming yeast work because screening only about a dozen yeast transformants is typically enough to find a clone with the region of interest. TAR cloning of chromosomal fragments, individual genes, or gene families can be used for functional, structural, and population studies, for comparative genomics, and for long‐range haplotyping, and has potential for gene therapy. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of CRISPR/Cas9‐treated genomic DNA for TAR cloning Basic Protocol 2: Isolation of a gene or genomic locus by TAR cloning Basic Protocol 3: Transfer of TAR/YAC/BAC isolates from yeast to E. coli