A CRISPR-guided mutagenic DNA polymerase strategy for the detection of antibiotic-resistant mutations in M. tuberculosis

Abstract

A sharp increase in multidrug-resistant tuberculosis (MDR-TB) threatens human health. Spontaneous mutation in essential gene confers an ability of Mycobacterium tuberculosis resistance to anti-TB drugs. However, conventional laboratory strategies for identification and prediction of the mutations in this slowly growing species remain challenging. Here, by combining XCas9 nickase and the error-prone DNA polymerase A from M. tuberculosis, we constructed a CRISPR-guided DNA polymerase system, CAMPER, for effective site-directed mutagenesis of drug-target genes in mycobacteria. CAMPER was able to generate mutagenesis of all nucleotides at user-defined loci, and its bidirectional mutagenesis at nick sites allowed editing windows with lengths up to 80 nucleotides. Mutagenesis of drug-targeted genes in Mycobacterium smegmatis and M. tuberculosis with this system significantly increased the fraction of the antibiotic-resistant bacterial population to a level approximately 60- to 120-fold higher than that in unedited cells. Moreover, this strategy could facilitate the discovery of the mutation conferring antibiotic resistance and enable a rapid verification of the growth phenotype-mutation genotype association. Our data demonstrate that CAMPER facilitates targeted mutagenesis of genomic loci and thus may be useful for broad functions such as resistance prediction and development of novel TB therapies.

Keywords: CRISPR; Mycobacterium tuberculosis; drug resistance; fitness; high-throughput sequencing; mutation.

Graphical abstract

Figure 1

Editing efficiency of XCas9 H840A/D10A combining with PolA3M^TB The mutagenesis tool CAMPER consisting of XCas9 nickase (nXCas9) and error-prone DNA polymerase A from Mtb (PolA3M^TB) was applied in Mycobacterium. (A) The CAMPER system constructed with XCas9 nickase and error-prone DNA polymerase A from M. tuberculosis nicked the target locus under the specific guidance of a gRNA and performed bidirectional nick translation for mutagenesis. (B) M. tuberculosis error-prone DNA polymerase A (PolA3M^TB) fused with nCas9 (Streptococcus pyogenes Cas9 containing a single point mutation, D10A) and SpCas9 (S. pyogenes Cas9). Compared with the off-target control, only the expression of PolA3M^TB fused with nCas9 led to an increase in bleomycin-resistant CFUs. (C) Evaluation of editing efficiency of CAMPER toward the chromosomal rpsL gene in M. smegmatis. A 100-μL electroporation culture was subcultured in 7H9 medium for mutant frequency calculations. (D) CAMPER for expression of XCas9 nickase H840A or XCas9 nickase D10A, under specific guidance of a gRNA, resulted in an increased rate of bleomycin-resistant cells compared with that in the off-target control. (E) Expression of CAMPER in M. smegmatis was induced with ATc at 0, 10, 50, or 200 ng/mL, and the transcriptional level was determined at 24 h with qPCR. The transcriptional level of MATURETB was highest under induction with 200 ng/mL ATc. (F) With the induction of 200 ng/mL ATc, the transcriptional level of CAMPER in M. smegmatis was measured after 8, 24, 48, and 96 h, which was highest at 24 h but decreased at 48 h. (G–I) Growth curves were measured to evaluate the cytotoxicity of CAMPER H840A/D10A with or without ATc induction. The growth curves were measured every 4 h, and the cell density at each time point was presented as the value of OD₆₀₀. (J) The consequences of nXCas9-PolA3MTB expression were monitored by spotting dilutions of each culture on LB agar. All of the above experiments were performed at least three times with similar results. Error bars indicate SEM for three biological replicates. A two-tailed unpaired t test was performed to determine the statistical significance of the data. ns, no significant difference; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 2

Construction of a CRISPR-guided DNA polymerase system in mycobacteria (A) Schematic diagram of the mutant frequency calculation assay workflow for calculation of the ratio of the resistant mutant versus the viable population. (B) High-throughput sequencing showing that CAMPER with an on-target gRNA resulted in random substitution mutation within ∼40-bp windows at both the 3′ side and 5′ side of the nick. The zero point represents the site nicked by CAMPER. (C and D) Two plasmids harboring bleo^R with an early stop codon were generated to verify the bidirectional editing feature of CAMPER. One stop codon, W23∗, was introduced at the 3′ side of the nick, requiring 3′-to-5′ nick translation for repair (C). Another E77∗ was introduced at the 5′ end of the nick, requiring 5' to 3′ nick translation for repair (D). Expression of gRNA-bleo^R 1 and 2, compared with off-target gRNA, both increased the bleomycin-resistant CFUs. (E) The editing efficiency of CAMPER in E. coli was assessed. We constructed a plasmid harboring bleoR with an early stop codon, B68∗. One gRNA targeting at the 5' side of the stop codon was designed (CAMPER 1), requiring 5′-to-3′ nick translation for repair. Another gRNA targeting at the 3′ side of the stop codon was also designed (CAMPER 2), requiring 3′-to-5′ nick translation for repair. Expression of gRNA CAMPER 1 and 2, compared with off-target gRNA, both increased the bleomycin-resistant CFUs. In addition, an EvolvR system was applied to repair the same stop codon from 5′ side to 3′ side, and the editing efficiency was similar to that of the CAMPER in E. coli. (F) Distribution of the nucleotides for bleo^R with an early stop codon (left) and mutated bleo^R repaired by CAMPER (right). All substitutions were derived from three independent biological replicates with the target strand as reference. All of the above experiments were performed at least three times with similar results. Error bars indicate SEM for three biological replicates. A two-tailed unpaired t test was performed to determine the statistical significance of the data. ns, no significant difference; ∗p < 0.1; ∗∗p < 0.01.

Figure 3

CAMPER enabled mutagenesis of target genes in mycobacteria (A) A guide targeting bleo^R with an early stop codon from plasmid was expressed in M. smegmatis. We subcultured 1, 10, 100, or 1,000 μL of electroporation culture in 7H9 medium for mutant frequency calculation assays. The percentage of bleomycin-resistant mutants per viable CFU was calculated. (B) A guide targeting chromosomal rpoB gene was expressed in M. smegmatis. We subcultured 1, 10, 100, or 1,000 μL of electroporation culture in 7H9 medium for mutant frequency calculation assays. The percentage of rifampicin-resistant mutants per viable CFU was calculated. For the assessment of CAMPER gene-editing efficiency in M. tuberculosis, four gRNAs were targeted to chromosomal rpsL (C and D) and atpE (E) from H37Ra, and rpsL (F) and atpE (G) from H37Rv. All of the above experiments were performed at least three times with similar results, and each dot in the bar graph represents one repeat. N.D, not detected. Error bars indicate SEM for all the repeats. A two-tailed unpaired t test was performed to determine the statistical significance of the data. ns, no significant difference; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 4

Characterization of drug-resistance-related mutations with CAMPER A total of six gRNAs were designed to identify the bedaquiline resistance-related mutations in atpE in M. smegmatis. The interaction between AtpE and bedaquiline is shown in (A). The region targeted by each gRNA and corresponding mutation frequency is shown in (B), where the mutation frequency of each region was determined with CFU assays. With the expression of gRNAs-215F, CAMPER increased the ratio of bedaquiline-resistant cells among the whole viable population. The locations of the mutations are shown in (C), which are labeled with red and the color shaded according to the mutation frequency of each region. Three biological replicates were tested. N.D, not detected. A total of 31 gRNAs were designed to identify the rifampicin resistance-related mutations in rpoB. The interaction between RpoB and rifampicin is shown in (D). The region targeted by each gRNA and corresponding mutation frequency are shown in (E), where the mutation frequency of each region was determined with CFU assays. The regions targeted by gRNA-3, gRNA-8, gRNA-9, gRNA-11, gRNA-12, and gRNA-16 displayed significantly higher mutation frequencies than that of the off-target control. The locations of the mutations are shown in (F), which are labeled with red and the color shaded according to the mutation frequency of each region. All of the above experiments were performed at least three times with similar results.

Figure 5

Using CAMPER to rapidly evolve rpoB RRDR of M. smegmatis (A) Schematic illustration of the competitive assay workflow for determining the fitness landscape of RRDR. pCAMPER and pGuider were both transformed into M. smegmatis. With the expression of gRNAs for target editing, CAMPER performed random substitution mutation among the 81-bp loci of RRDR. During cultivation, the system mutated the target region continuously, owing to induction with ATc. Mutagenesis accumulated among the entire viable population. After the saturation stage, all cultures were plated on agar containing rifampicin to screen out rifampicin-resistant (RIF^R) mutants, and were considered as the T0 library. For competitive assays, equal amounts of RIF^R mutants were subcultured in 7H9 medium containing rifampicin, and cultures without rifampicin were used as a control. Samples were collected at T1, and the 81-bp DNA fragment of RRDR was amplified via PCR. Genotypes and related frequencies were further verified via Illumina HiSeq sequencing. The fitness of each genotype was evaluated as the increase in frequency under antibiotic selection relative to that of wild-type RRDR. (B) The number of genotypes changed under treatment with different concentrations of rifampicin. (C) Surface representation of the M. smegmatis RNA polymerase β subunit and steric hindrance of rifampicin binding to the RRDR. The RRDR is in blue, and the region with mutations detected at T0 is in pink. (D) Fitness landscape of RRDR under treatment with rifampicin at 0, 80, or 160 μg/mL at T1. Each tile represents a variant with one single-base substitution (x axis) at one specific position (y axis), whose relative growth is indicated by the color of the tile scale according to the corresponding color scale bar on top. In addition, mutants with a frequency <0.06% are marked with a black dot. (E) Drug susceptibility comparison of pool-selected and reconstructed strains for rifampicin-resistant mutants D516N and H526Y (MIC fold change RIF^R/parent strain). Each dot in the bar graph represents one repeat. (F) Growth curve pattern comparison among pool-selected and reconstructed strains for the rifampicin-resistant mutants D516N and H526Y. The cell density is presented as the value of OD₆₀₀ and was measured every 4 h. All of the above experiments were performed at least three times with similar results. Error bars indicate SEM for all the repeats.