Abstract
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.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune systems are widely distributed among almost all archaea and a large number of bacteria, protecting microbes against invasion by foreign DNAs, such as viruses [1]
The expression of ddCpf1 was driven by a constitutive BioBrick promoter of J23100 in a plasmid containing p15A ori, while the crRNA transcription was driven by the BioBrick promoter of J23109 in a colE1 plasmid
The above results demonstrated the repression of lacZ transcription was conducted by the ddCpf1/crRNA complex, and a proper protospacer-adjacent motif (PAM) sequence was necessary for the repression
Summary
The clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune systems are widely distributed among almost all archaea and a large number of bacteria, protecting microbes against invasion by foreign DNAs, such as viruses [1]. Besides of Cas, another CRISPR-Cas protein Cpf, which belongs to the class 2 type V-A CRISPR-Cas system, is widely applied for genome editing in many organisms [21,22,23,24,25,26,27,28]. Cpf is so far the most minimalistic CRISPR-Cas systems with dual DNase and RNase activities [29]. Its dual activities have been employed to process a single customized CRISPR array with its RNase activity and cut target DNAs with its DNase activity, allowing for multiplex genome editing in both mammalian cells and rice [30, 31]. Because multiple mature crRNA can be conveniently obtained, the system can be applied for convenient multiplex genome editing
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