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A Novel Eukaryote‐Like CRISPR Activation Tool in Bacteria

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A Novel Eukaryote‐Like CRISPR Activation Tool in Bacteria METHODS, MODELS & TECHNIQUES Prospects & Overviews www.bioessays-journal.com A Novel Eukaryote-Like CRISPR Activation Tool in Bacteria: Features and Capabilities Yang Liu and Baojun Wang* friend or foe” system, scientists are CRISPR (clustered regularly interspaced short palindromic repeats) activation able to guide the endonucleases to (CRISPRa) in bacteria is an attractive method for programmable gene their desired DNA or RNA targets.[5–8] activation. Recently, a eukaryote-like, 54-dependent CRISPRa system has CRISPR regulation relies on inactivated been reported. It exhibits high dynamic ranges and permits flexible target site CRISPR endonucleases. The nuclease- deficient CRISPR DNA endonucleases, selection. Here, an overview of the existing strategies of CRISPRa in bacteria for instances dCas9 and ddCpf1 (dCas12), is presented, and the characteristics and design principles of the CRISPRa are effectively programmable DNA bind- system are introduced. Possible scenarios for applying the eukaryote-like ing domains. When these domains are CRISPRa system is discussed with corresponding suggestions for tethered to transactivation domains or performance optimization and future functional expansion. The authors subunits of RNA polymerase, they ac- envision the new eukaryote-like CRISPRa system enabling novel designs in tivate the promoters near the CRISPR target sites. This strategy has been 54 multiplexed gene regulation and promoting research in the -dependent widely utilized in both eukaryotes and gene regulatory networks among a variety of biotechnology relevant or prokaryotes,[9–17] particularly in the former, disease-associated bacterial species. where the transcription activation mecha- nisms and the activators are well-studied. While CRISPRa in eukaryotes enjoys much success and is continuously im- 1. Introduction proved, the development of CRISPRa in prokaryotes had stagnated.[10,15,16] Bikard et al. reported the CRISPR (clustered regularly interspaced short palindromic re- first bacterial CRISPRa device to employ an RNA polymerase peats) activation (CRISPRa) is a power and versatile technology -subunit fused dCas9 in a -deleted strain.[10] The second for genetic engineering and biological research. The innate pro- CRISPRa device has an E. coli activator SoxS, which was recruited grammability from the CRISPR module offers unprecedented to the dCas9 through the engineered single guide RNA (sgRNA) flexibility to turn on any target gene of interest. Its programmabil- and an RNA-binding domain fused to the activator. SoxS inter- ity was instrumental to CRISPR, which is an adaptive immunity acts with the -subunit of RNA polymerase. This enhances the in bacteria.[1–3] Prokaryotes have evolved mechanisms to protect activation efficacy and removes the prerequisite on host strain themselves against exogenous DNA/RNA. They were performed genetic backgrounds.[15] Recently, a dCas9-fused anti-sigma fac- by endonucleases that recognize the invasive species through tor AsiA was employed for a CRISPRa system, which enriched RNA/DNA or RNA/RNA complementary pairing and then cleav- the bacterial CRISPRa toolbox.[17] ing them.[4,5] By exploiting the sequence-specific “identification The above introduced bacterial CRISPRa systems are designed for bacterial 70-dependent genes, which perform most house- keeping functions.[18] CRISPRa systems for many other genes were still unavailable, for instance, many other biological func- Y.Liu,Dr.B.Wang School of Biological Sciences tions that respond to environmental changes under the control University of Edinburgh of the 54 factor, the only sigma factor apart from 70 responsible Edinburgh EH9 3FF, UK for regulating various functions.[19] The 54-dependent promot- E-mail: [email protected] ers have a distinct activation mechanism from its 70 counterpart, Y.Liu,Dr.B.Wang with a unique promoter structure and its own set of conserved Centre for Synthetic and Systems Biology [20] University of Edinburgh core sequences. Their regulations work over long distances Edinburgh EH9 3FF, UK with a DNA looping structure, similar to that of the RNA poly- merase II in eukaryotes. Hence, 54 activation is also known as The ORCID identification number(s) for the author(s) of this article eukaryote-like gene activation in bacteria.[21–25] Much of the regu- can be found under https://doi.org/10.1002/bies.201900252 latory networks and biological functions of 54-dependent genes © 2020 The Authors. BioEssays published by WILEY Periodicals, Inc. This remain elusive, a situation that hindered the standardization and is an open access article under the terms of the Creative Commons application of 54-dependent promoters in genetic engineering. Attribution License, which permits use, distribution and reproduction in 54 any medium, provided the original work is properly cited. Recently, we developed a CRISPRa system for -dependent genes. It supports high dynamic range regulation with low DOI: 10.1002/bies.201900252 BioEssays 2020, 42, 1900252 1900252 (1 of 10) © 2020 The Authors. BioEssays published by WILEY Periodicals, Inc. www.advancedsciencenews.com www.bioessays-journal.com Table 1. Comparison between existing bacterial CRISPRa systems. Cas protein Regulator Activator attachment mode Target promoters Host cell background requirement CRISPR/dCas9 -subunit Protein fusion/gRNA scaffold mediated 70 promoter E. coli ΔrpoZ[10] CRISPR/dCas9 SoxS gRNA scaffold mediated 70/38/32/24 promoter None in E. coli[15] CRISPR/dCas9 AsiA (including mutant) Protein fusion/gRNA scaffold mediated 70 promoter None in E. coli, K. oxytoca, S. enterica (for protein fusion) & 70 F563Y mutated E. coli (for gRNA scaffold mediated)[15,17] CRISPR/dCas9 PspFΔHTH gRNA scaffold mediated 54 promoter None in E. coli, K. oxytoca, E. coli ΔpspF (for NorR enhanced function),[16] WtsA CRISPR/dCas9 factors Protein fusion 70 promoter None in M. xanthus[26] CRISPR/dCas9 TetD gRNA scaffold mediated 70 promoter None in E. coli[15] CRISPR/dCas9 - DNA looping 70/54 promoter None in E. coli[27] expression leakiness.[16] Thanks to the flexible DNA looping separable domains are independently responsible for DNA bind- and the inherently long-distance regulation, CRISPRa target ing, regulation, and recruitment of RNA polymerase or other sites can be placed over a wider physical range. Furthermore, transcription factors. These domains can be mixed and matched, it enables direct activation of many 54-dependent promoters, and would trigger transcription activation as long as they are which otherwise have to be activated by environmental stim- fused or assembled together to bring the RNA polymerase and ac- uli that would have global effects and complicate experimen- cessory transcription factors to the vicinity of the promoter. This tal control. These properties of our CRISPRa, together with our simple and yet robust characteristic is also the cornerstone of the discoveries of CRISPRa design principles, will facilitate the re- two-hybrid system, which screens for molecular interactions in search in 54-dependent gene regulatory networks and the ap- vivo: Candidate proteins/domains are fused to a DNA binding plications of 54-dependent promoters in synthetic biology and domain and an activation domain respectively. Any interaction industry. between candidates would recruit the activation domain to the We illustrated the application of the 54-dependent CRISPRa DNA binding domain and activate the promoter nearby, which by two examples. We built a two-layered cascaded activation and converts interaction into observable gene expression. a positive feedback regulation. These circuits demonstrated the The sufficiency of an activator-RNA polymerase interaction potential of our CRIPSRa to build complex gene regulatory net- to initiate bacterial transcription was first reported in 1997. It works. We also used a standardized 54 promoter library to im- was simultaneously the first reported bacterial two-hybrid (B2H) plement a high throughput method for screening the multi-gene system that utilized the -subunit of the RNA polymerase.[28,29] expression profile. A multi-gRNA generator circuit was designed This was followed by Dove and Hochschild in 1998, who showed to optimize a multi-gene pathway by projecting various transcrip- that the -subunit of the RNA holoenzyme could also be used tion profiles onto it. We envision that the success of our CRISPRa to recruit and stabilize the RNA polymerase to enhance tran- design and its application would open up exciting opportuni- scription initiation.[30] This system, however, only works when ties, and encourage other scientists to apply the eukaryote-like the endogenous copy of the -subunit (rpoZ) has been knocked CRISPRa system for their scientific endeavors. out. The -subunit is an important component in RNA holoen- zyme, which can respond to the alarmone ppGpp during strin- gent response and broadly regulate gene transcription. However, 2. Existing CRISPR Activation Methods in Bacteria the -subunit (rpoZ) is not essential and its knockout does not To date, three canonical CRISPRa systems and one gene acti- significantly affect the E. coli host growth rate. Hence the func- vation device using CRISPR-mediated DNA looping have been tion of this subunit was once vague and controversial for a long [31–33] proven in bacteria. Here we briefly summarize their designs, time. The interaction between the -subunit and the other properties, and their suitable applications (Table 1). The sum- subunits of RNA polymerase is thought to contribute to its ability mary, therefore, is an
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