Gene silencing with CRISPRi in bacteria and optimization of dCas9 expression levels.

TLDR: This article describes a method to optimize the expression level of dCas9 in order to avoid toxicity while ensuring strong on-target repression activity and demonstrates this method by optimizing a pLZ12 based vector originally developed for S. aureus so that it can work in E. coli.

About: This article is published in Methods.The article was published on 2020-02-01 and is currently open access. It has received 31 citations till now. The article focuses on the topics: Cas9 & CRISPR.

Figures

Fig. 3.1) is required to block the elongating RNA polymerase [14,15,27]. 3. The repression efficiency was initially reported to be inversely correlated with the target

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Gene silencing with crispri in bacteria and optimization of dcas9 expression levels" ?

Here the authors provide detailed design rules, methods and novel vectors to perform CRISPRi experiments in S. aureus and in E. coli, using the well characterized dCas9 protein from S. pyogenes. In particular, the authors describe a vector based on plasmid pC194 which is broadly used in Firmicutes, as well as a vector based on the very broad host-range rolling circle plasmid pLZ12, reported to replicate in both Firmicutes and Proteobacteria. The authors describe a method to optimize the expression level of dCas9 in order to avoid toxicity while ensuring strong on-target repression activity. The authors demonstrate this method by optimizing a pLZ12 based vector originally developed for S. aureus so that it can work in E. coli. This article should provide all the resources required to perform CRISPRi experiments in a broad range of bacterial species. A potential caveat of adapting dCas9 tools to various bacterial species is that dCas9 was shown to be toxic when expressed too strongly. 

Q2. What type of systems have been the exploited?

Type II systems, and in particular the one from Streptococcus pyogenes, have been the most exploited since they rely on a single protein, Cas9, for target DNA cleavage [18,19]. 

Q3. How can a plasmid be used to perform inducible CRISPRi?

good clones can be used to perform inducible CRISPRi experiments by growing them on a medium with or without anhydrotetracycline (aTc). 

Q4. What was used as host for the plasmids?

E. coli E4644 (TransforMax™ EC100D™ pir+, Lucigen) was used as host for recombinant plasmids based on pOSIP-KL and was grown at 30°C in LB broth or agar supplemented with kanamycin 50 µg/ml. 

Q5. What is the common variant of Cas9 used to silence genes?

When dCas9 is coexpressed with a guide RNA, it forms a DNA recognition complex that binds to the specific target, like wild-type Cas9, but without cleavage of the DNA. 

Q6. What is the PCR method used to confirm the plasmid?

The first PCR enables to identify clones carrying the guide sequence and the second PCR to amplify a fragment which can be used to confirm the guide sequence by Sanger sequencing. 

Q7. How many clones are screened for the first PCR?

The authors typically obtain six good clones out of eight screened colonies for the first PCR, and Sanger sequencing results show a mutation only once every ten screened clones on average. 

Q8. What is the strand orientation of dCas9 binding?

The strand orientation of dCas9 binding does not seem to matter when blocking transcriptioninitiation by targeting a promoter sequence; however when targeting inside a transcribed region, binding of the guide RNA to the non-template (coding) strand (for example, primer F205,fitness defect produced when targeting downstream of essential genes, and not through direct measurement of RNA levels. 

Q9. How do the authors control gene expression in bacteria?

The authors provide simple yet powerful methods, to control gene expression in bacteria by using dCas9 to block transcription initiation or elongation. 

Q10. What is the way to avoid the introduction of extra nucleotides during the assembly?

To avoid the introduction of extra nucleotides during the assembly of the specific guide sequence in their plasmids, the authors use the type IIS BsaI restriction enzyme which cleaves outside of its recognition sequence (Fig. 2 and Fig. 3.3). 

Q11. What is the way to test dCas9 in E. coli?

This strain allows, in one step, the screening of clones able to survive the expression of dCas9 in the presence of a bad seed sequence, while still efficiently silencing the expression of mCherry. 

Q12. What is the effect of dCas9 on the chromosome?

This phenomenon called the “bad-seed” effect is particularly observed at high dCas9 concentrations, and could be decreased by tuning dCas9 levels while maintaining strong on-target repression. 

Q13. What are the advantages of using silencing techniques?

Gene silencing techniques can be inducible, enabling the study of essential genes, and are frequently easier to implement than targeted mutagenesis techniques. 

Q14. What is the effect of the empty vector on plating efficiency in S. aureus 8?

Effect of the empty vector, a non targeting sgRNA control, the T-rpsL guide targeting the template strand or the C-rpsL guide targeting the coding strand on plating efficiency in S. aureus 8235-4. 

Q15. What was the effect of pFD116 on mCherry?

As expected, guides programmed to target the S. aureus rpsL gene had no effect on E. coli (Fig. 7).Finally, the authors evaluated the ability of pFD116 and pFD152 to block the expression of a targetmCherry gene without toxicity as a function of aTc concentration. 

Q16. Why is the PAM not present in the sgRNA?

It is important to note that the PAM needs to be present immediately downstream of the targetsite in the genomic DNA, but the NGG sequence is not introduced in the sgRNA (Fig. 3.2).