FLASH-TB: an Application of Next-Generation CRISPR to Detect Drug Resistant Tuberculosis from Direct Sputum

Abstract

Offering patients with tuberculosis (TB) an optimal and timely treatment regimen depends on the rapid detection of Mycobacterium tuberculosis (Mtb) drug resistance from clinical samples. Finding Low Abundance Sequences by Hybridization (FLASH) is a technique that harnesses the efficiency, specificity, and flexibility of the Cas9 enzyme to enrich targeted sequences. Here, we used FLASH to amplify 52 candidate genes probably associated with resistance to first- and second-line drugs in the Mtb reference strain (H37Rv), then detect drug resistance mutations in cultured Mtb isolates, and in sputum samples. 92% of H37Rv reads mapped to Mtb targets, with 97.8% of target regions covered at a depth ≥ 10X. Among cultured isolates, FLASH-TB detected the same 17 drug resistance mutations as whole genome sequencing (WGS) did, but with much greater depth. Among the 16 sputum samples, FLASH-TB increased recovery of Mtb DNA compared with WGS (from 1.4% [IQR 0.5-7.5] to 33% [IQR 4.6-66.3]) and average depth reads of targets (from 6.3 [IQR 3.8-10.5] to 1991 [IQR 254.4-3623.7]). FLASH-TB identified Mtb complex in all 16 samples based on IS1081 and IS6110 copies. Drug resistance predictions for 15/16 (93.7%) clinical samples were highly concordant with phenotypic DST for isoniazid, rifampicin, amikacin, and kanamycin [15/15 (100%)], ethambutol [12/15 (80%)] and moxifloxacin [14/15 (93.3%)]. These results highlighted the potential of FLASH-TB for detecting Mtb drug resistance from sputum samples.

Keywords: CRISPR; FLASH; Mycobacterium tuberculosis; RNA guide; drug resistance; sequencing.

FIG 1

Data quality of FLASH-TB on DNA extract from lab strain H37Rv. (A) Proportion of reads mapped or unmapped to 50 targeted genes. (B) Average depth and (C) Coverage of 50 targeted genes at depth ≥ 10X. (A to C) Five nanogram DNA input. (D) Proportion of reads mapped to 50 targeted genes and their coverage at different amounts of DNA input. The results were presented as mean ± SD from triplicated experiments.

FIG 2

Depth and coverage of individual gene targets included in WHO tier 1 catalogue from FLASH-TB on DNA extract from lab strain H37Rv from independent triplicated experiments. (A and C) Proportion of different depth levels across the individual gene. (B and D) Depth of each single nucleotide across the entire individual gene was plotted. Each line represented for an independent experiment. The blue dots indicated possible mutations associated with drug resistance from Mykrobe catalogue. The red dots indicated the position of guide RNAs where CRISPR/Cas9 targets to cleave. (A and B) Thirteen genes known to be associated with drug resistance from Mykrobe catalogue. (C and D) genes not listed in Mykrobe catalogue, embABC: embA-embB-embC.

FIG 3

FLASH-TB and dWGS on DNA extracts from sputum of 16 TB patients with different levels of bacterial load determined by Zeihl-Neilsen staining, ranging from AFB (acid-fast bacilli) 1+ to 3+. (A) Proportion of TB reads. (B) Average depth of reads mapped to 50 gene targets. (C) Coverage of 50 targeted genes at depth >3. Gold for dWGS, blue for FLASH-TB from direct sputum.