Efficient Transcriptional Gene Repression by Type V-A CRISPR-Cpf1 from Eubacterium eligens

Abstract

Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is an emerging technology for artificial gene regulation. Type II CRISPR-Cas endonuclease Cas9 is the most widely used protein for gene regulation with CRISPRi. Here, we present type V-A CRISPR-Cas endonuclease Cpf1-based CRISPRi. We constructed an l-rhamnose-inducible CRISPRi system with DNase-deactivated Cpf1 from Eubacterium eligens (EedCpf1) and compared its performance with catalytically deactivated Cas9 from Streptococcus pyogenes (SpdCas9). In contrast to SpdCas9, EedCpf1 showed stronger gene repression when it was targeted to the template strand than when it was targeted to the nontemplate strand of the 5' untranslated region or coding DNA sequences. EedCpf1 exhibited no strand bias when targeted to the promoter, and preferentially used the 5'-TTTV-3' (V = A, G, or C) protospacer adjacent motif. Multiplex repression of the EedCpf1-based CRISPRi system was demonstrated using episomal and chromosomal gene targets. Our findings will guide an efficient EedCpf1-mediated CRISPRi genetic control.

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

“…Besides dFnCpf1 and dLbCpf1, dCpf1s from Acidaminococcus sp. and Eubacterium eligens have also been tested in bacterial and eukaryotic cells (17)(18)(19)(20). Our screening and prediction methods could serve a pipeline facilitating the transformation of these proteins into powerful tools for diverse application of multiplex gene 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).…”

Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors

“…Recently described CRISPR-Cpf1 nucleases offer additional capabilities beyond those of SpCas9 including shorter length CRISPR RNAs (crRNAs) for guiding Cpf1 to targets, the ability to target T-rich PAMs [11][12][13] , and RNase processing of multiple crRNAs from a single transcript by sequence-specific ribonuclease activity resident within Cpf1 itself 14,15 . However, to our knowledge, "dead" Cpf1-based gene regulators have thus far only been shown to repress gene expression in bacteria 16,17 and plant (Arabidopsis) 18 and have not been shown to function in mammalian cells either as activators or repressors. Here we describe both constitutively active and chemically inducible Cpf1-based transcriptional activator platforms for upregulating human gene promoters.…”

Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding

“…Mature CRISPR RNAs (crRNAs) derived from the CRISPR array can in turn program Cas nucleases to recognize and cleave DNA targets whose nucleic acid sequence is complementary with the guide portion of the crRNA and proximal to a PAM (protospacer adjacent motif) site. Due to their simple and programmable nature, the nucleases of class 2 CRISPR systems, particularly Cas9 (type II) and Cas12 (type V), have been the subject of intense research interest for the purposes of genome editing [2][3][4], programmable gene regulation utilizing a catalyticallydead CRISPR nuclease (dCas) [5][6][7], and nucleic acid detection [8,9].…”