Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination.
Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination.
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DOI：
10.1038/s41467-021-21275-4
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发表时间：
2021-02-19
影响因子：
16.6
通讯作者：
Zhao H
中科院分区：
文献类型：
--
作者：
Enghiad B;Huang C;Guo F;Jiang G;Wang B;Tabatabaei SK;Martin TA;Zhao H
直接克隆是获取大量未表征的天然产物生物合成基因簇（BGC）以发现新的生物活性化合物的最有效策略。然而，由于它们的大尺寸，重复性或高GC含量，这些BGC的大规模克隆仍然是一个巨大的挑战。在这里，我们报告了一种可扩展的直接克隆方法，称为Cas 12 a辅助的精确靶向克隆，使用体内Cre-lox重组（CAPTURE），该方法包括Cas 12 a消化，称为T4聚合酶exo + fill-in DNA组装的DNA组装方法，以及Cre-lox体内DNA环化。我们应用这种方法克隆了47个BGC，从10到113 kb的放线菌和芽孢杆菌与~100%的效率。克隆的BGC的异源表达导致发现了15种先前未表征的天然产物，包括具有独特的5/6/6/6/5五环碳骨架的六种环状头-尾异二聚体，命名为bipentaromycins A-F。四种双戊霉素对革兰氏阳性和革兰氏阴性细菌如耐甲氧西林金黄色葡萄球菌、耐万古霉素屎肠球菌和生物武器炭疽芽孢杆菌均显示出较强的抗微生物活性。由于其鲁棒性和效率，我们的直接克隆方法加上异源表达提供了一个有效的策略，大规模发现新的天然产物。由于生物合成基因簇的大小和重复性，直接克隆它们是困难的。在这里，作者介绍了CAPTURE，它使用Cas 12 a和体内Cre-lox重组来有效克隆高达113 kb的基因簇，并展示了这种方法如何用于大规模发现新的天然产物。
Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A–F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products. Direct cloning of biosynthetic gene clusters is difficult due to their size and repetitive nature. Here, the authors present CAPTURE, which uses Cas12a and in vivo Cre-lox recombination for efficient cloning of gene clusters up to 113 kb, and demonstrate how this method can be used for large-scale discovery of novel natural products.
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影响因子：
14.9
作者：
Lei C;Li SY;Liu JK;Zheng X;Zhao GP;Wang J
通讯作者：
Wang J
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
3.2
作者：
Bunet, Robert;Song, Lijiang;Aigle, Bertrand
通讯作者：
Aigle, Bertrand
通过构建生产者细菌人工染色体文库异源表达阿维菌素生物合成基因簇。
影响因子：
4.8
作者：
Deng Q;Zhou L;Luo M;Deng Z;Zhao C
通讯作者：
Zhao C
影响因子：
14.8
作者：
Kouprina, Natalay;Noskov, Vladimir N.;Larionov, Vladimir
通讯作者：
Larionov, Vladimir