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. 2017 Jun 6;3:17018.
doi: 10.1038/celldisc.2017.18. eCollection 2017.

Multiplex gene regulation by CRISPR-ddCpf1

Xiaochun Zhang  1   2   3 , Jingman Wang  4   5 , Qiuxiang Cheng  6 , Xuan Zheng  1 , Guoping Zhao  1 , Jin Wang  1
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Multiplex gene regulation by CRISPR-ddCpf1

Xiaochun Zhang et al. Cell Discov. .
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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.

Keywords: CRISPR; CRISPRi; Cpf1; DNase-dead Cpf1 (ddCpf1); multiplex gene regulation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Programmable gene repression by ddCpf1/crRNA. (a) ddCpf1-mediated repression of the transcription lacZ in MG1655. Positions of the crRNAs and sgRNAs designed for lacZ were illustrated, targeting to either the T strand or the NT strand of lacZ. Although only the crRNAs targeting to the T strand showed remarkable repression. The transcription of lacZ in cells expressing ddCpf1 alone was employed as a control, and the value was normalized to 1000. Two sgRNAs were also designed, and the dCas9-mediated repression was more effective with sgRNA targeting to the NT strand, which could be found in Supplementary Figure S1. (b) ddCpf1-mediated repression of the transcription malT in MG1655. crRNAs were designed to target both the promoter region and the T strand in the coding region of malT, and the guide sequences could be found in Supplementary Table S1. The transcription of malT in cells expressing ddCpf1 only was employed as a control, and the value was normalized to 1 000. Symbols of malT-T, malT-TP and malT-NTP represented crRNAs targeting to the T strand in the coding region, T strand in the promoter region and the NT strand in the promoter region of malT gene, respectively.
Figure 2
A proposed model for ddCpf1-mediated repression of gene transcription. The ddCpf1/crRNA complex bound to the T strand in the coding sequence of a target gene, blocking the transcription elongation of the RNAP. Alternatively, the ddCpf1/crRNA complex could bind to the promoter region of its target gene to block the transcription initiation, which was not shown in this model.
Figure 3
Whole-transcriptome RNA-seq analysis of the specificity of ddCpf1-mediated repression. Cells expressing ddCpf1 with or without lacZ-T1 crRNA were analyzed, and according to the FPKM values, only the transcription of the lac operon was remarkably repressed, demonstrating the high specificity of ddCpf1-mediated repression. Genes of ddAsCpf1, lacZ, lacY and LacA were highlighted.
Figure 4
Multiplex gene repression with ddCpf1 and a crRNA array. Four target genes (malT, proP, degP and rseA) were analyzed, employing the non-target rpoE gene as an internal control. The order of the target genes was shown, which differed in array 1 from array 2. The transcriptional level of each gene was analyzed in cells expressing ddCpf1 with either individual crRNA or crRNA arrays, and cells expressing ddCpf1 only were employed as a control. For rpoE gene, its transcriptional level was analyzed in cells individually expressing all the tested crRNAs, and its value in cells expressing ddCpf1 only was normalized to 1000.
Figure 5
Prompt characterization of TCSs in E. coli with the ddCpf1-mediated multiplex gene repression strategy. The 32 RRs in E. coli were divided into 6 groups (Supplementary Tables S3 and S4), and the growth phenotypes were analyzed by culture in either rich LB medium (a) or minimal M9 medium (b). In c, cells expressing ddCpf1 and individual crRNAs that targeted genes in arrayG5 were cultured in M9 medium, employing arrayG5 as a control. The growth-deficient strains, which expressed ddCpf1 and arrayG5 (or crRNAs targeting genes in arrayG5), were marked with the red dashed box, while the yellow dashed box marked the slow growth phenotype of arrayG6. A gradient dilution of cells with sterile water was performed, and different amounts of cells (that is, 4.0×105, 4.0×104, 4.0×103, 4.0×102 and 4.0×101) were dotted on plates. Cells expressing ddCpf1 only were employed as the positive control.
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