Multiplex gene regulation by CRISPR-ddCpf1

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.

Systematically investigating the key features of the nuclease deactivated Cpf1 for tunable multiplex genetic regulation

“…Next, we sought to demonstrate the ability of multiplex gene regulation of the CRISPR-dCpf1 system. As the targeting of multiple genes has been demonstrated in several recent studies (17,18,20), and a single bound dCpf1, without dedicated inactivation domains, was not sufficient in suppressing gene expression in human HEK293T cells (20), we studied gene repression by tandemly positioned dCpf1 roadblocks within a single gene. Guide sequences were selected to target three independent segments within the coding region of the sf-gfp gene.…”

“…Although aspects of the CRISPR-Cpf1 system as DNA endonuclease has been characterized, there have been only first attempts in using CRISPR-dCpf1 as transcriptional regulators. These studies proved its applicability in bacterial, plant, and mammalian cells (17)(18)(19)(20). To harness and streamline the system for multiplex gene regulation, three specific aspects need addressing or systematic characterization: 1. a mutational scheme that abolishes Cpf1's nuclease activity and yet minimally affects its DNA binding and RNase activities; 2. the requirements for pre-crRNA that contains multiple direct repeat-guide sequence units for efficient crRNA processing and DNA targeting (14,21); and 3. the dependence of DNA binding strength on the PAM sequence (22)(23)(24)(25).…”

Impact of CRISPR interference on strain development in biotechnology

“…Cas12a is so far the smallest CRISPR system with dual DNase and RNase activity [45]. Interestingly, a DNase‐dead version of Cas12a with the amino acid exchange E993A termed ddCas12a (or ddCpf1) [46] preserved the RNase activity, but lacked DNAse activity. Therefore, ddCas12a in theory can be employed to process its precursor crRNA as well as a customized CRISPR array [45,46].…”

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit

“…Recent studies demonstrated that DNase-dead Cas12a can also be used for gene regulation in bacteria and plants (Tang et al, 2017; Zhang et al, 2017), suggesting that a wider range of applications will also be enabled by dCas12a.…”