Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9.
Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9.
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DOI：
10.1042/bsr20203595
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发表时间：
2021-01-29
影响因子：
4
通讯作者：
Bolt EL
中科院分区：
文献类型：
--
作者：
Lau CH;Bolt EL
CRISPR 系统通过 Cas1-Cas2 蛋白复合物催化的 DNA 捕获和整合，建立针对移动遗传元件的适应性免疫。最近的研究表明，CRISPR 重复序列和适应模块起源于一种新型 DNA 转座子，称为 casposon。 Casposons 编码一种称为 Casposase 的 Cas1 同源物，它单独整合到含有来自 Casposons 的末端反向重复序列 (TIR) 的靶分子单链和双链 DNA 中。最近的一项研究表明，Methanosarcina mazei casposase 能够整合随机 DNA 寡核苷酸，在这项工作中使用 Acidoprofundum boonei casposase，我们还观察到混杂的底物整合。在这里，我们首先表明，Acidoprofundum boonei caspase 的底物灵活性扩展到无需 TIR 的 DNA 随机整合，包括功能基因的整合。然后，我们用它来研究 Casposase 催化的 DNA 整合反应对特定 DNA 位点的靶向，从而允许插入定义的 DNA 有效负载。 Casposase-Cas9 融合体经过工程设计，在体外具有良好的催化能力，可以从短的合成 DNA 或基因（带或不带 TIR）生成 RNA 引导的 DNA 整合产物。然而，由于 caspase 部分的竞争背景活性，DNA 整合仍然可能在不受引导的情况下发生。 Casposase-dCas9 在大肠杆菌细胞中的表达有效地靶向染色体和质粒 lacZ，这表明β-半乳糖苷酶活性降低，但未检测到 DNA 整合。 Casposase 的混杂底物整合特性使其成为潜在的 DNA 插入工具。 Casposase-dCas9 融合蛋白可以作为独立于同源定向 DNA 修复的 DNA 插入基因编辑开发的原型。
CRISPR systems build adaptive immunity against mobile genetic elements by DNA capture and integration catalysed by Cas1–Cas2 protein complexes. Recent studies suggested that CRISPR repeats and adaptation module originated from a novel type of DNA transposons called casposons. Casposons encode a Cas1 homologue called casposase that alone integrates into target molecules single and double-stranded DNA containing terminal inverted repeats (TIRs) from casposons. A recent study showed Methanosarcina mazei casposase is able to integrate random DNA oligonucleotides, followed up in this work using Acidoprofundum boonei casposase, from which we also observe promiscuous substrate integration. Here we first show that the substrate flexibility of Acidoprofundum boonei casposase extends to random integration of DNA without TIRs, including integration of a functional gene. We then used this to investigate targeting of the casposase-catalysed DNA integration reactions to specific DNA sites that would allow insertion of defined DNA payloads. Casposase–Cas9 fusions were engineered that were catalytically proficient in vitro and generated RNA-guided DNA integration products from short synthetic DNA or a gene, with or without TIRs. However, DNA integration could still occur unguided due to the competing background activity of the casposase moiety. Expression of Casposase-dCas9 in Escherichia coli cells effectively targeted chromosomal and plasmid lacZ revealed by reduced β-galactosidase activity but DNA integration was not detected. The promiscuous substrate integration properties of casposases make them potential DNA insertion tools. The Casposase–dCas9 fusion protein may serves as a prototype for development in genetic editing for DNA insertion that is independent of homology-directed DNA repair.