Efficient genome editing in pathogenic mycobacteria using Streptococcus thermophilus CRISPR1-Cas9

Abstract

The ability to genetically engineer pathogenic mycobacteria has increased significantly over the last decades due to the generation of new molecular tools. Recently, the application of the Streptococcus pyogenes and the Streptococcus thermophilus CRISPR-Cas9 systems in mycobacteria has enabled gene editing and efficient CRISPR interference-mediated transcriptional regulation. Here, we converted CRISPR interference into an efficient genome editing tool for mycobacteria. We demonstrate that the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) is functional in Mycobacterium marinum and Mycobacterium tuberculosis, enabling highly efficient and precise DNA breaks and indel formation, without any off-target effects. In addition, with dual sgRNAs this system can be used to generate two indels simultaneously or to create specific deletions. The ability to use the power of the CRISPR-Cas9-mediated gene editing toolbox in M. tuberculosis with a single step will accelerate research into this deadly pathogen.

Keywords: CRISPR-Cas9 system; Genome editing; Indels; Mycobacterium marinum; Mycobacterium tuberculosis.

Figure 1. Gene editing using CRISPR-Cas9 in M. marinum.

(A) Schematic overview of CRISPR-Cas9-mediated gene editing. Following induction with ATc, Sth1Cas9 and the designed sgRNA are expressed and form a complex. Following the recognition of the target site next to the PAM, the endonuclease domains of Sth1Cas9 will induce a double-stranded DNA break which can result in bacterial killing if not repaired. NHEJ results in error-prone DNA repair, which can lead to frameshift mutations and subsequent knockout of the target gene. (B) Survival of M. marinum E11 transformants carrying pCRISPRx-Sth1Cas9-L5 containing sgRNA-crtb-1. Survival was calculated as the percentage of the total number of colonies compared to the number of colonies carrying the control plasmid. Bars represent the mean values and standard deviations from two experiments. (C-E) Percentages of phenotypically white colonies using sgRNA-crtb-1 in M. marinum E11 (C) and M (D) strains, and sgRNA-crtb-2 in M. marinum M (E). Percentages of phenotypically white colonies were calculated as the percentage of white colonies in proportion to the total number of colonies. Mean and standard deviation are shown from two replicate experiments and three replicate experiments for sgRNA-crtb-1 in M. marinum M. (F) Frequency of indel mutants for several genes in M. marinum E11. Separate colonies with or without ATc treatment were screened for indels using target amplification and sequencing analysis. The number of colonies harboring indels at the target site are shown in white, and WT colonies are shown in gray. (G) Number of colonies containing CRISPR-Sth1Cas9-induced mutations for several genes tested in M. marinum M as identified by sequencing. Separate colonies were subjected to genome target amplification and sequencing analysis. WT colonies are shown in gray, and mutant colonies are shown in white. (H) Frequency of mutation events observed for all mutated colonies in M. marinum E11 and M strains as determined by PCR and subsequent sequencing analysis for the indicated genes.

Figure 2. CRISPR-Cas9 mediated gene editing in M. tuberculosis.

(A) The number of colonies harboring indel mutations in M. tuberculosis CDC1551. Transformants carrying the pCRISPRx-Sth1Cas9-L5 containing sgRNA targeting either PE_PGRS16 (MT1004), pecA (MT2036) or PE26 (MT2595) were induced on 7H10 plates containing ATc for 3 weeks. Separate colonies were tested for mutations at the sgRNA target sites using PCR and sequencing. WT colonies are shown in gray, and mutant colonies are shown in white. (B) The number of colonies containing indel mutations in M. tuberculosis H37Rv. The pCRISPRx-Sth1Cas9-L5 containing sgRNA targeting either katG (Rv1908c), PE_PGRS16 (Rv0977), pecA (Rv1983) or PE26 (Rv2519) were electroporated in M. tuberculosis H37Rv. Genome editing was induced for 3 weeks on 7H10 plates supplemented with ATc. Mutants were identified using target amplification and sequencing. Colonies identified as WT are shown in gray, and mutant colonies are shown in white. (C) Frequency and type of indels observed at sgRNA target sites among the mutants detected. (D) Isoniazid (INH)-resistance of katG frameshift mutants. Two WT and katG frameshift mutant strains were spotted on 7H10 plates containing different concentrations of INH. Colony density was measured using ImageJ analysis. Data for each strain was normalized to the untreated condition.

Figure 3. CRISPR-Cas9-mediated gene deletions using dual sgRNAs in M. marinum.

(A) Schematic overview of CRISPR-Cas9-induced gene deletions using dual sgRNAs. Mycobacteria carrying the pCRISPRx-Sth1Cas9-L5 plasmid encoding two sgRNAs will express Sth1Cas9 and dual sgRNAs upon treatment with ATc. The sgRNAs will direct the Sth1Cas9 enzymes towards the designed genomic target regions. Target recognition will lead to the induction of double-stranded DNA breaks which can result in the generation of single indels, double indels, or accurate deletion events upon distinct repair events. Extended deletions beyond the 20 bp sgRNA target site give rise to inaccurate deletions. (B) Percentage of phenotypic crtB mutants identified as white colonies of untreated or ATc-induced M. marinum M using sgRNA-crtb-1 and sgRNA-crtb-2 simultaneously. The percentage phenotypic crtB mutants was calculated as the percentage of white colonies in proportion to the total number of colonies. The mean and standard deviation is shown from two replicate experiments. (C) Percentage of indel and deletion events using dual sgRNAs. White colonies obtained with or without ATc treatment were subjected to PCR and sequencing analysis. Percentages were calculated from the total sequenced colonies per condition (untreated n=19, 1h ATc n=16, 10 days ATc n=13).