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Multiplex gene regulation by CRISPR-ddCpf1

机译：CRISPR-ddCpf1的多重基因调控

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The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli , and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9. When targeting the promoter region, both strands showed effective repression by the ddCpf1/crRNA complex. The whole-transcriptome RNA-seq technique was further employed to demonstrate the high specificity of ddCpf1-mediated repression. Besides, we proved that the remaining RNase activity in ddCpf1 was capable of processing a precursor CRISPR array to simply generate multiple mature crRNAs in vivo , facilitating multiplex gene regulation. With the employment of this multiplex gene regulation strategy, we also showed how to quickly screen a library of candidate targets, that is, the two-component systems in E. coli . Therefore, based on our findings here, the CRISPR-ddCpf1 system may be further developed and widely applied in both biological research and clinical studies.
机译：簇状规则间隔的短回文重复序列（CRISPR）/ dCas9系统已广泛应用于转录调控和表观遗传学研究。然而，对于多个靶标，需要多个单个指导RNA（sgRNA）的独立表达，这不太方便。为了解决这个问题，我们采用了DNase-dead Cpf1突变体（ddCpf1）进行多重基因调控。我们证明了ddCpf1可以单独用于大肠杆菌中的基因阻遏，并且使用CRISPR RNA（crRNA）特异性靶向其靶基因的模板链更有效，这与dCas9不同。当靶向启动子区域时，两条链均受到ddCpf1 / crRNA复合物的有效抑制。进一步采用全转录组RNA-seq技术来证明ddCpf1介导的阻遏的高特异性。此外，我们证明了ddCpf1中剩余的RNase活性能够加工CRISPR前体阵列，从而在体内简单地产生多个成熟的crRNA，从而促进了多重基因调控。通过使用这种多重基因调控策略，我们还展示了如何快速筛选候选靶标库，即大肠杆菌中的两组分系统。因此，基于我们在这里的发现，CRISPR-ddCpf1系统可能会得到进一步开发，并广泛应用于生物学研究和临床研究。

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