Construction Of CRISPR Interference(CRISPRi) System And Its Application In Essential Gene Function Of Mycobacteria

Part one Construction of CRISPR Interference?CRISPRi?System in MycobacteriaObjective Despite technical advances in introducing genomic deletions and modulating gene expression,direct inactivation of essential genes in mycobacteria remains difficult.In this study,we used the pkn B as a target gene to construct a CRISPR?clustered regularly interspaced short palindromic repeats?interference system in Mycobacterium smegmatis?Msm?.This study is the first research to construct this novel CRISPRi approach in mycobacteria in our country.Meanwhile,we established a detailed experimental approach on the critical steps required to obtain high quality RNA extracts in mycobacteria.We also showed the experimental procedure on how to use the 3':5' integrity assay to measure RNA integrity for quantitative assessment in mycobacteria.This study laid the technic groundwork for functional studies of essential gene in mycobacteria and treatment or prevention in tuberculosis.Methods Plasmids?p RH2502 and p RH2521?used in this study were obtained from Addgene.p RH2502 was a mycobacterial integrating vector expressing an inactive version of Cas9?CRISPR-associated protein 9?.p RH2521 was a replicating vector in which the sg RNA?single-guide RNA?was expressed.p RH2502 was introduced into M.smegmatis using electroporation.M.smegmatis strains harboring p RH2502 were supplemented with or without a Tc?anhydrotetracycline?and cultures were grown for 12 h.Cells from M.smegmatis cultures were harvested at 0 h and 12 h for RNA isolation.RNA quality?purity and integrity?was determined by analysis of the A260/280 ratio following spectrophotometry,the ribosomal RNA bands on agarose gels and the reference gene sig A 3':5' integrity assay by quantitative PCR.The expression of dcas9 was determined by RT-q PCR?reverse transcription quantitative polymerase chain reaction?.Growth curves of cultures in M.smegmatis containing dcas9 were constructed to measure the influence of overexpression of dcas9?dead cas9?on bacterial growth.The phosphorylated ds DNA?double-stranded DNA?insert that contained the gene-specific sg RNA sequence was then ligated with the linearized and dephosphorylated p RH2521 vector.p RH2521 vector was cut at the Bbs I site,phosphatase treated and then purified using a gel extraction kit.Two oligonucleotides containing 20 bases homologous to the pkn B sequence plus 4 bases complementary to the overhangs of the digested p RH2521,were synthesized,annealed and phosphorylated.The phosphorylated ds DNA insert that contained the gene-specific sg RNA sequence was then ligated with the linearized and dephosphorylated p RH2521 vector.The ligated material was used to transform Escherichia coli DH5? and recombinants harboring p RH2521 with gene-specific sg RNA sequences were confirmed by sequencing.Finally,p RH2521 with the gene-specific sg RNA sequence was transformed into M.smegmatis that already contained p PR2502.M.smegmatis strains harboring both p RH2502 and p RH2521 were supplemented with or without a Tc and cultures were grown for 12 h.The RNA was extracted and purified as above.The expressions of dcas9 and pkn B were determined by RT-q PCR.Growth curves of cultures were constructed to measure the influence of depleted Msm_pkn B on bacterial growth.The measurements for expressions were depicted as the mean±SD?standard deviation?which obtained from five independent experiments to correct the trial errors.The statistical significance of the differences between the expressions of a Tc-induced versus uninduced cultures was determined using Two-Way ANOVA?Prism 6.0,Graph Pad software?.Results?1?We demonstrated that this CRISPRi?CRISPR interference?approach could successfully induce overexpression of dcas9 and silence pkn B in M.smegmatis.For the induced M.smegmatis strain expressing dcas9 and sg RNAs targeting pkn B,271.4±75.3-fold induced expression of dcas9 and 3.94±1.76-fold decreased expression of pkn B were observed,along with 74% reduced expression of pkn B,and the differences were statistically significant between a Tc-12 h group and N-a Tc-0h group.Additionally,overexpression of dcas9 did not affect the bacterial growth of M.smegmatis.The depleted pkn B did not result in obvious growth phenotype change, which may need an extended growth curves for a more definitive conclusion.?2?The A260/280 ratios of all the RNA samples used for RT-q PCR were in the range of 1.8–2.0.The brightness ratios of 23S/16 S on agarose gels were approximately 2.0,with a low number of short fragments.All of these results confirmed that the input materials were of high purity and integrity.?3?Moreover,the 3':5' ratios of all the RNA samples used for RT-q PCR were in the range of 1.1–3.6.The result was considered to indicate good RNA integrity.Conclusion?1?This study established a CRISPR interference system in mycobacteria,and we demonstrated that this CRISPRi approach could successfully induce overexpression of dcas9 and silence pkn B in M.smegmatis.The CRISPRi approach is extremely promising for application in the repression of essential genes of unknown function in mycobacteria.?2?This study provided detailed information on the critical steps required to obtain high quality RNA extracts,including the use of TE buffer containing lysozyme and proteinase K,hot QIAzol lysis reagent,mechanical disruption,purification and contaminating DNA removing.?3?This study provided experimental procedure of 3':5' integrity assay to measure RNA integrity in mycobacteria.This method supports the reliability and validity of data on the measurement of genetic expression.Part two Application of CRISPRi System in Essential Gene Function of MycobacteriaObjective This study is the first research to investigate the function of an essential gene involving Msm_F by repressing gene expression using the novel CRISPRi approach.First,this study used the CRISPRi approach to repress Msm_F expression,and observed the growth and morphology changes in depleted Msm_F cells.Then we were able to test the gene essentiality of Msm_F during cell division.Second,the expressions of the corresponding off-target genes were measured to prove the low off-target effects of the CRISPRi system.Third,the expressions of the two interacting partners?Msm_fts Z and Msm_mur G?were measured to reveal the mechanism mediating the interaction between cell division proteins.Meanwhile,a simple and adaptable method was established to accomplish the CRISPRi target site selection and identification of off-target effects.Methods The sg RNAs binding to the antisense strand of the coding region were designed against the 20 nt sequence after the PAM?protospacer-adjacent motif?sequence ‘5'-CCN-3''closer to the 5' end of the gene-coding region.The specificity of sg RNA binding in the M.smegmatis genome was checked using the CAS OT search tool.Three sg RNAs lacking homology with other sequences in the M.smegmatis genome were selected.The positions of the selected sg RNAs relative to the translation start codon were-1,+46 and +101.For the construction of a F-specific p RH2521 plasmid,two complementary oligonucleotides were synthesized and annealed together,yielding a 24-bp DNA fragment with GGGA and CAAA overhangs at the two ends,respectively.The annealed ds DNA was cloned at the Bbs I site into p RH2521 as described in the part one.Three F-specific p RH2521 vectors were constructed in this study,including Msm_F-1,Msm_F+46 and Msm_F+101.Moreover,a potato nucleic acid sequence of 101 bp lacking homology with any M.smegmatis sequence,was used to design a control sg RNA. Then p RH2521 with the gene-specific and control sg RNA sequences were transformed into M.smegmatis that already contained p PR2502.M.smegmatis strains harboring both p RH2502 and p RH2521 were supplemented with or without a Tc and cultures were grown for 12 h.The RNA was extracted and purified as above.The expressions of dcas9,F,off-target genes and two interacting partners?Msm_fts Z and Msm_mur G?were determined by RT-q PCR.Growth curves of cultures were constructed to measure the influence of depleted Msm_F on bacterial growth.Microscopy of cells was proceeded to study the the cell morphology resulting from CRISPRi on Msm_F.The measurements for expressions were depicted as the mean±SD which obtained from 3-4 independent experiments to correct the trial errors.The statistical significance of the differences between the expressions of a Tc-induced versus uninduced cultures was determined using Two-Way ANOVA?Prism 6.0,Graph Pad software?.Results?1?We demonstrated that this CRISPRi approach could successfully induce overexpression of dcas9 and silence F in M.smegmatis.For the induced M.smegmatis strain expressing dcas9 and sg RNAs targeting the-1,+46 and +101 sequences,180.0±43.7,262.0±60.7 and 208.3±36.4-fold induced expression of dcas9,60.7±11.9,18.8±1.7 and 38.3±8.3-fold decreased expression of F were observed,along with 98%,95% and 97% reduced expression of F,respectively.The differences were statistically significant between a Tc-12 h group and N-a Tc-0h group.An induced expression of dcas9 was observed in the strain containing the sg RNA control vector,while no decrease in expression of F was observed.?2?The depleted Msm_F resulted in growth and morphology changes.For each induced strain,decreased growth was observed from 9 h following induction;large amounts of dead bacterial cells were observed at 24 h.A significant proportion of the bacteria exhibited elongation,filamentation and branch-like structures at 12 h after a Tc induction.Direct observation of the cell length distribution revealed that the majority of cells were at least 2.5-fold longer than the mean cell length of strains in culture without a Tc addition. ?3?The expressions of the four off-target genes were measured and no significant reduction in expression was observed for any of these genes.Compared with N-a Tc-0h group,0.86±0.21,0.91±0.15,0.82±0.18 and 0.87±0.14-fold expression of MSMEG₆529,MSMEG₅505,MSMEG₁378 and MSMEG₅373 were observed in the strains of Msm_dcas9+F-1,Msm_dcas9+F+46 and Msm_dcas9+F+101 in a Tc-12 h group,respectively.?4?The expressions of the two interacting partners?Msm_fts Z and Msm_mur G?were measured and the levels of Msm_fts Z and Msm_mur G were not affected by the absence of Msm_F.Compared with N-a Tc-0h group,0.93±0.09 and 0.96±0.09-fold expression of Msm_fts Z and Msm_mur G were observed in the strain of Msm_dcas9+F-1 in a Tc-12 h group.?5?Considering the CRISPRi target site selection,all three of the designed sg RNAs in this study were effective at repressing the target gene,giving a success rate of 100%.Considering the identification of off-target effects,the CAS OT tool was a simple and efficient tool for checking the specificity of sg RNA binding in the M.smegmatis genome.?6?Moreover,a potato nucleic acid sequence of 101 bp?SPUD sequence?,lacking homology with any mycobacterial sequence by using the CAS OT tool,was first used to design a control sg RNA in this study.The potential off-target DNA binding sites of less than 5 mismatches with the whole mycobacterial genome were only 0-1 of all the four designed sg RNAs.This potato sequence was suitable for designing the sg RNA control vector lacked homology to any mycobacterial sequence.Conclusion?1?This study demonstrated that the novel CRISPRi approach could successfully repress the expression of essential gene Msm_F in physiological level.?2?This study demonstrated that the depleted Msm_F could result in growth and morphology changes.These findings indicated that F plays an important role in coordinating cell division in M.smegmatis.?3?The expression levels of the four off-target genes demonstrated that the CRISPRi system could specifically repress a target gene with minimal off-target effects.?4?The expression levels of the two interacting partners indicated that Msm_F may interact on a structural rather than a regulatory level with Msm_fts Z and Msm_mur G.?5?Considering the CRISPRi target site selection,this study suggested multiple sg RNAs binding to the antisense strand of the 5' end coding region was a good choice.Considering the identification of off-target effects,we concluded that the CAS OT tool was especially suitable for species with no professional website.?6?This study first used a SPUD sequence to design a control sg RNA vector that targeted no gene-specific sequence in mycobacteria.This study demonstrated that the SPUD sequence had low homology with mycobacterial sequence?both Mtb and Msm?using the CAS OT tool.