A multiplex CRISPR interference tool for virulence gene interrogation in an intracellular pathogen

Ellis, N.; Kim, Byoungkwan; Machner, Matthias P.

doi:10.1101/2020.06.17.157628

Cited by 3 publications

(2 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Lastly, the potential for Rho termination should be considered for biotechnological applications that require arrays with multiple spacers. Indeed, a recent study showed that including a boxA upstream of a 10-spacer CRISPR array substantially improves the efficiency of multiplexed CRISPRi in Legionella pneumophila ( Ellis et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

Transcription termination and antitermination of bacterial CRISPR arrays

Stringer

Baniulyte

Lasek‐Nesselquist

et al. 2020

eLife

View full text Add to dashboard Cite

A hallmark of CRISPR-Cas immunity systems is the CRISPR array, a genomic locus consisting of short, repeated sequences ('repeats') interspersed with short, variable sequences ('spacers'). CRISPR arrays are transcribed and processed into individual CRISPR RNAs that each include a single spacer, and direct Cas proteins to complementary sequence in invading nucleic acid. Most bacterial CRISPR array transcripts are unusually long for untranslated RNA, suggesting the existence of mechanisms to prevent premature transcription termination by Rho, a conserved bacterial transcription termination factor that rapidly terminates untranslated RNA. We show that Rho can prematurely terminate transcription of bacterial CRISPR arrays, and we identify a widespread antitermination mechanism that antagonizes Rho to facilitate complete transcription of CRISPR arrays. Thus, our data highlight the importance of transcription termination and antitermination in the evolution of bacterial CRISPR-Cas systems.

show abstract

Section: Discussionmentioning

confidence: 99%

Transcription termination and antitermination of bacterial CRISPR arrays

Stringer

Baniulyte

Lasek‐Nesselquist

et al. 2020

eLife

View full text Add to dashboard Cite

show abstract

“…Further, knockdown gradients can be achieved by expressing CRISPRi components from constitutive promoters of differing strengths (Qu et al., 2019), by using truncated spacers (Vigouroux, Oldewurtel, Cui, Bikard, & Teeffelen, 2018), or by systematically mutating the sgRNA to introduce mismatches between the spacer and target gene that reduce knockdown efficacy to a predictable extent (Hawkins et al., 2020). Finally, CRISPRi can be multiplexed to target several genes in the same cell by cloning arrays of sgRNAs with different spacers (Ellis, Kim, & Machner, 2020; Peters et al., 2016; Reis et al., 2019). These advantages suggest that CRISPRi will become a common tool for bacterial functional genomics in the near future.…”

Section: Introductionmentioning

confidence: 99%

Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

et al. 2020

View full text Add to dashboard Cite

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, Tn7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICEBs1 transfer 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: Tn7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4: ICEBs1 transfer 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 of E. coli by electroporation Support Protocol 4: Transformation of CaCl2‐competent E. coli

show abstract

Transcription Termination and Antitermination of Bacterial CRISPR Arrays

Stringer

Baniulyte

Lasek‐Nesselquist

et al. 2018

Preprint

View full text Add to dashboard Cite

A hallmark of CRISPR-Cas immunity systems is the CRISPR array, a genomic locus consisting of short, repeated sequences (“repeats”) interspersed with short, variable sequences (“spacers”). CRISPR arrays are transcribed and processed into individual CRISPR RNAs (crRNAs) that each include a single spacer, and direct Cas proteins to complementary sequence in invading nucleic acid. Most bacterial CRISPR array transcripts are unusually long for untranslated RNA, suggesting the existence of mechanisms to prevent premature transcription termination by Rho, a conserved bacterial transcription termination factor that rapidly terminates untranslated RNA. We show that CRISPR arrays of Salmonella Typhimurium are protected from Rho by the Nus factor anti-termination complex, and we provide evidence that this is an evolutionarily ancient mechanism to facilitate complete transcription of bacterial CRISPR arrays.

show abstract

A multiplex CRISPR interference tool for virulence gene interrogation in an intracellular pathogen

Cited by 3 publications

References 58 publications

Transcription termination and antitermination of bacterial CRISPR arrays

Transcription termination and antitermination of bacterial CRISPR arrays

Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

Transcription Termination and Antitermination of Bacterial CRISPR Arrays

Contact Info

Product

Resources

About