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基因组编辑揭示了 pSCL4 在. 中维持染色体线性所必需的。

Genome editing reveals that pSCL4 is required for chromosome linearity in .

机构信息

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

Present address: Department of Bioresources for Bioeconomy and Health Research, Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany.

出版信息

Microb Genom. 2021 Nov;7(11). doi: 10.1099/mgen.0.000669.

DOI:10.1099/mgen.0.000669
PMID:34747689
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8743545/
Abstract

is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid's gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

摘要

是一种具有工业重要性的放线菌，其遗传操作受到低转化和接合效率、引入 DNA 重组水平低以及难以获得一致孢子形成的限制。我们描述了用于该菌株 Cas9 介导的基因组编辑的多功能载体的构建和应用。为了有信心地设计间隔序列，我们对该模式株（ATCC 27064）的一个分离株进行了高度准确的基因组组装。这产生了一个染色体组装（6.75 Mb）加上 pSCL4（1795 kb）和 pSCL2（149 kb）的组装。该菌株还携带 pSCL1（12 kb），但由于其体积小，只有部分序列被覆盖。以前描述的 pSCL3（444 kb）不在该分离株中。使用我们的 Cas9 载体，我们通过靶向质粒的 基因高效地消除了 pSCL4。其中五个产生的 pSCL4 消除的分离株被表征，所有分离株的孢子形成都受到损害。对每个这些衍生物的 shotgun 基因组测序显示，它们的染色体末端都有大片段缺失，对于两个克隆，获得了足够的序列数据表明染色体已经环化。这些数据表明，pSCL4 对线性染色体的结构稳定性是必不可少的。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f58/8743545/94fb49d9aa2f/mgen-7-0669-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f58/8743545/1052139d039c/mgen-7-0669-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f58/8743545/f0e585840904/mgen-7-0669-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f58/8743545/94fb49d9aa2f/mgen-7-0669-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f58/8743545/1052139d039c/mgen-7-0669-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f58/8743545/f0e585840904/mgen-7-0669-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f58/8743545/94fb49d9aa2f/mgen-7-0669-g003.jpg

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