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Title: Regulated Expression of sgRNAs Tunes CRISPRi in E. coli
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NSF-PAR ID:
10061220
Author(s) / Creator(s):
 ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Biotechnology Journal
Volume:
13
Issue:
9
ISSN:
1860-6768
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Abstract  
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  2. Abstract

    Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn7transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICEBs1transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors.

    Basic Protocol 1: sgRNA design

    Basic Protocol 2: Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors

    Basic Protocol 3: Tn7transfer of Mobile‐CRISPRi to Gram‐negative bacteria

    Basic Protocol 4: ICEBs1transfer of Mobile‐CRISPRi to Bacillales

    Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters

    Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates

    Support Protocol 3: Transformation ofE. coliby electroporation

    Support Protocol 4: Transformation of CaCl2‐competentE. coli

     
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  3. Abstract

    CRISPRi-mediated gene regulation allows simultaneous control of many genes. However, highly specific sgRNA-promoter binding is, alone, insufficient to achieve independent transcriptional regulation of multiple targets. Indeed, due to competition for dCas9, the repression ability of one sgRNA changes significantly when another sgRNA becomes expressed. To solve this problem and decouple sgRNA-mediated regulatory paths, we create a dCas9 concentration regulator that implements negative feedback on dCas9 level. This allows any sgRNA to maintain an approximately constant dose-response curve, independent of other sgRNAs. We demonstrate the regulator performance on both single-stage and layered CRISPRi-based genetic circuits, zeroing competition effects of up to 15-fold changes in circuit I/O response encountered without the dCas9 regulator. The dCas9 regulator decouples sgRNA-mediated regulatory paths, enabling concurrent and independent regulation of multiple genes. This allows predictable composition of CRISPRi-based genetic modules, which is essential in the design of larger scale synthetic genetic circuits.

     
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  4. Premise

    Light is critical in the ability of plants to accumulate chlorophyll. When exposed to far‐red (FR) light and then grown in white light in the absence of sucrose, wild‐type seedlings fail to green in a response known as theFRblock of greening (BOG). This response is controlled by phytochrome A through repression of protochlorophyllide reductase‐encoding (POR) genes byFRlight coupled with irreversible plastid damage. Sigma (SIG) factors are nuclear‐encoded proteins that contribute to plant greening and plastid development through regulating gene transcription in chloroplasts and impacting retrograde signaling from the plastid to nucleus.SIGs are regulated by phytochromes, and the expression of someSIGfactors is reduced in phytochrome mutant lines, including phyA. Given the association of phyA with theFR BOGand its regulation ofSIGfactors, we investigated the potential regulatory role ofSIGfactors in theFR BOGresponse.

    Methods

    We examinedFR BOGresponses insigmutants, phytochrome‐deficient lines, and mutant lines for several phy‐associated factors. We quantified chlorophyll levels and examined expression of keyBOG‐associated genes.

    Results

    Among sixsigmutants, only thesig6 mutant significantly accumulated chlorophyll afterFR BOGtreatment, similar to thephyAmutant.SIG6 appears to control protochlorophyllide accumulation by contributing to the regulation of tetrapyrrole biosynthesis associated with glutamyl‐tRNAreductase (HEMA1) function, select phytochrome‐interacting factor genes (PIF4andPIF6), andPENTA1, which regulatesPORAmRNAtranslation afterFRexposure.

    Conclusions

    Regulation ofSIG6plays a significant role in plant responses toFRexposure during theBOGresponse.

     
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  5. Atomi, Haruyuki (Ed.)
    ABSTRACT CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N 2 ) fixation were targeted for dCas9-mediated repression. First, the nif operon ( nifHI 1 I 2 DKEN ) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD . Remarkably, growth of M. acetivorans with N 2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N 2 . Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1 , encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N 2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans . IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens. 
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