首页> 外文期刊>Biology Direct >Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells
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Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells

机译:Cpf1核酸酶显示出强大的活性,可通过利用哺乳动物细胞中的同源性定向修复途径来诱导DNA修饰

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Background Cpf1 nucleases have recently been repurposed for site-specific genome modification. Two members of the Cpf1 family, the AsCpf1 from Acidaminococcus sp. and the LbCpf1 from Lachnospiraceae bacterium were shown to induce higher indel frequencies than SpCas9 when examining four randomly-selected target sequences for each type of nuclease. Whether they are a real match for Cas9 nucleases, however, remains to be verified. Results Here, we used AsCpf1 and LbCpf1 to induce homology directed repair, either single strand annealing (SSA) or homologous recombination (HR), in N2a mouse neuroblastoma cells. Exploiting a plasmid that contains two GFP halves with overlapping sequences and exploring 20 targets, on all but one both nucleases consistently performed with above 10?% efficiency. Several Cas9 nucleases have been previously characterised in order to find an orthogonal counterpart for the most widely used promiscuous SpCas9. Here, we found that AsCpf1 and LbCpf1 might be better candidates than three of the best such counterparts: Cas9 from Staphylococcus aureus , from Streptococcus thermophilus and from Neisseria meningitidis , when assessed for inducing efficient SSA mediated repair in N2a cells. When tested on genomic targets exploiting HR, both nucleases were able to induce the integration of a donor cassette with 1000?bp-long homologous arms. We also generated plasmids that express these Cpf1 nucleases together with their cognate crRNAs and that are equipped with type IIS restriction enzyme sites to facilitate spacer cloning. Conclusions Our results suggest that employing As- or LbCpf1 nuclease to induce homology directed repair in N2a cells, although is less effective at present than employing SpCas9, it is an equally or more effective tool than the most frequently used orthogonal Cas9 counterparts of SpCas9. These findings support the position of Cpf1 nucleases on the side of SpCas9 on the palette of effective genome engineering tools. Reviewers This article was reviewed by Eugene Koonin, Haruhiko Siomi and Jean-Yves Masson.
机译:背景Cpf1核酸酶最近已被重新用于位点特异性基因组修饰。 Cpf1家族的两个成员,来自Acidaminococcus sp。的AsCpf1。当检查每种类型的核酸酶的四个随机选择的靶序列时,Lachnospiraceae细菌的LbCpf1和LbCpf1比SpCas9诱导更高的indel频率。但是,它们是否与Cas9核酸酶真正匹配尚待验证。结果在这里,我们使用AsCpf1和LbCpf1在N2a小鼠神经母细胞瘤细胞中诱导同源性定向修复,无论是单链退火(SSA)还是同源重组(HR)。利用一个质粒,该质粒包含两个具有重叠序列的GFP片段,并在除了两个核酸酶以外的所有核酸酶上以20%以上的效率一致地进行工作,并探索20个靶标。先前已经对几种Cas9核酸酶进行了表征,以便找到最广泛使用的混杂SpCas9的正交对应物。在这里,我们发现,AsCpf1和LbCpf1可能比三个最佳的类似物更好:候选金黄色葡萄球菌,嗜热链球菌和脑膜炎奈瑟氏球菌的Cas9,在评估它们在N2a细胞中诱导有效的SSA介导的修复时。当对利用HR的基因组靶标进行测试时,两种核酸酶均能够诱导供体盒与1000 bp长的同源臂整合。我们还生成了表达这些Cpf1核酸酶及其同源crRNA的质粒,并配备了IIS型限制酶位点以促进间隔子克隆。结论我们的结果表明,使用As-或LbCpf1核酸酶在N2a细胞中诱导同源性定向修复,尽管目前效果不如使用SpCas9,但与SpCas9的最常用正交Cas9对应物相比,它是同等或更有效的工具。这些发现支持了Cpf1核酸酶在SpCas9一侧在有效基因组工程工具面板上的位置。审阅者本文由Eugene Koonin,Haruhiko Siomi和Jean-Yves Masson审阅。

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