Materials and Methods

A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing inMycobacterium tuberculosis

Strains, media, and growth conditions.

E. coli strain MG1655, M. marinum strain M, M. bovis BCG, M. tuberculosis strains H37Ra and H37Rv, M. smegmatis strain mc2155, and their derivatives were used in this study. Unless otherwise indicated, M. tuberculosis H37Ra was used. M. smegmatis was grown in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween 80 and 0.2% glycerol or on Middlebrook 7H10 plates supplemented with the appropriate antibiotics. M. marinum, M. bovis BCG, and M. tuberculosis were grown in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween 80, 0.2% glycerol, and oleic acid-albumin-dextrose-catalase (OADC; Becton Dickinson) or on 7H10 plates supplemented with the appropriate antibiotics, 0.5% glycerol, and OADC (Becton Dickinson). When indicated, antibiotics and small molecules were used at the following concentrations: kanamycin (25 μg/ml), hygromycin (50 μg/ml), zeocin (50 μg/ml), and anhydrotetracycline (ATc) (50 ng/ml).

Plasmids.

pMV261-Cas12a and pCR-Hyg (or pCR-Zeo) were used in M. marinum. The genes encoding the MmNHEJ machinery (MMAR_457_3, _MMAR_4574, and MMAR_4575) were amplified from the M. marinum chromosome. Plasmids containing cas9_Sth1 were originally obtained from pLJR965 (48). pNHEJ-Cas12a-_recX and pNHEJ-Cas12a-recA_mu (or pNHEJ-Cas9Sth1-_recX) were used in M. smegmatis, and pYC1655 was constructed to express the cognate sgRNA. pNHEJ-recX-sacB (or pNHEJ-recA_mu-_sacB) were used in M. tuberculosis. A codon-optimized cas9 gene under the control of the TetR-regulated promoter and the sgRNA cassette under the control of an optimized TetR-regulated promoter were cloned into a plasmid harboring the pMF1 replicon (51) to yield pYC1640 (or pYC2085). The sgRNA cassette contains two BbsI restriction sites for insertion of the target sequence. A recX allele was amplified from the E. coli chromosome and cloned into pZCas9 (23) to create pZCas9-recX. All plasmids used in this study are described in Table S4 in the supplemental material. Details of plasmid constructions are available upon request.

TABLE S4

Plasmids used in this study. Download Table S4, PDF file, 0.2 MB.

Copyright © 2020 Yan et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Genome editing in M. smegmatis.

Cells harboring pNHEJ-Cas12a-recX (or pNHEJ-Cas9Sth1-recX) were grown in 4 ml of 7H9 medium supplemented with 0.05% Tween 80, 0.2% glycerol, and 25 μg/ml kanamycin. One or two milliliters of starter culture was used to inoculate 100 ml complete 7H9 broth in a 250-ml flask, which was incubated at 37°C overnight. Competent M. smegmatis cells were prepared as previously described (59). About 300 ng of the crRNA-expressing plasmid was mixed with the competent cells. Electroporation was performed with the following settings: 2.5 kV, 25 μF, and 1,000-Ω resistance. After electroporation, 1 ml of 7H9 broth was added to the cells, which were then immediately incubated for 4 h at 30°C. Next, the cultures were plated on 7H10 agar supplemented with the appropriate antibiotics and 50 ng/ml ATc. When targeting the gfp gene, the editing efficiency was calculated as the frequency of GFP-negative (white) transformants. Each time, at least 24 GFP-negative colonies were picked for PCR and sequencing analysis. To cure the helper plasmids, a single colony was picked and grown to saturation in 7H9 medium at 37°C, followed by plating on 7H10 plates. The colonies from these plates were streaked to screen for loss of antibiotic resistance, and more than 70% of the colonies had lost the two helper plasmids in most cases.

Genome editing in M. marinum.

Cells harboring pMV261-Cas12a were grown in 4 ml of 7H9 medium supplemented with 0.05% Tween 80, 0.2% glycerol, OADC, and 25 μg/ml kanamycin. One or two milliliters of starter culture was used to inoculate 100 ml complete 7H9 broth in a 250-ml flask, which was incubated at 30°C for 3 to 5 days. Competent M. marinum cells were prepared as previously described (60). Electroporation of M. marinum was performed in a similar manner as for M. smegmatis. After electroporation, the cells were cultured in 1 ml of 7H9 broth supplemented with OADC and incubated overnight at 30°C. Next, the cultures were plated on 7H10 agar supplemented with OADC, the appropriate antibiotics, and 50 ng/ml ATc. The plates were incubated for 10 to 15 days at 30°C, after which time transformants were picked for PCR and sequencing analysis. Target-specific primers were designed to amplify sequences at least 1,000 bp upstream and downstream from the chromosomal sequences flanking the cleavage sites. The PCR products were analyzed via agarose gel electrophoresis, and the colonies with smaller or no PCR products were regarded as mutants. The PCR products amplified from the transformants with sizes similar to that of the wild-type strain were further analyzed via sequencing. The editing efficiency was calculated as the ratio of the number of edited events to the total number of colonies analyzed by PCR and sequencing. To cure the helper plasmids, a single colony was picked and grown to saturation in 7H9 medium supplemented with OADC at 30°C, followed by plating the cells on 7H10 plates supplemented with OADC. The colonies from these plates were streaked to screen for loss of antibiotic resistance, and more than 50% of the colonies had lost the two helper plasmids in most cases.

Genome editing in M. tuberculosis.

Cells harboring pNHEJ-recX-sacB (or pNHEJ-recA_mu-_sacB) were grown in 10 ml of 7H9 medium supplemented with 0.05% Tween 80, 0.2% glycerol, OADC, and 25 μg/ml kanamycin. One or two milliliters of starter culture was used to inoculate 100 ml complete 7H9 broth in a roller bottle, which was incubated at 37°C for 5 to 7 days. At an optical density at 600 nm (OD600) of ∼0.8, 10 ml of 15% sterilized glycine stock solution was added, yielding a final concentration of 1.5%, and the incubation continued at 37°C with rolling for an additional 20 to 24 h. Competent M. bovis BCG and M. tuberculosis cells were prepared as previously described (61). Electroporation of M. bovis BCG and M. tuberculosis was performed similarly to that of M. marinum and M. smegmatis with the following modifications. After electroporation, the cells were cultured in 1 ml of 7H9 broth supplemented with OADC and incubated for 2 days at 37°C to allow cells to grow at high density, mimicking the stationary-phase environment. Next, the cultures were plated on 7H10 agar supplemented with OADC, the appropriate antibiotics, and 50 ng/ml ATc. The plates were incubated for 20 to 30 days at 37°C, and transformants were then picked for PCR and sequencing analysis. To cure the helper plasmids, a single colony was picked and grown to saturation in 7H9 medium supplemented with OADC and 5 μg/ml kanamycin at 37°C. The cells were then diluted 1:100 in 1 ml of 7H9 medium supplemented with OADC and grown for 5 days. The cultures were diluted and plated on 7H10 plates supplemented with OADC and 2% sucrose. The colonies from these plates were streaked to screen for loss of antibiotic resistance, and more than 70% of the colonies had lost the two helper plasmids in most cases.

For the sequential mutation of four genes in M. tuberculosis, the Rv3408 mutant was first constructed as described above using a Zeor sgRNA-expressing plasmid. Next, a verified colony was picked to prepare competent cells, which were then transformed with a Hygr sgRNA-expressing plasmid targeting Rv0059. The transformants obtained were verified to have the Rv0059 mutation and loss of Zeocin resistance. A verified colony was picked to prepare competent cells, which were then transformed with a Zeor plasmid expressing double sgRNAs targeting Rv2494 and Rv2596 to simultaneously generate the double mutations, generating an M. tuberculosis strain with mutations in four genes.

Genome editing in E. coli.

Genome editing in E. coli was performed as described previously (23). Briefly, the competent E. coli cells harboring the appropriate plasmids were transformed with control or lacZ targeting the sgRNA plasmid and then plated on the appropriate selective LB plate supplemented with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) (40 μg/ml) and IPTG (isopropyl-β-d-thiogalactopyranoside) (100 μM). Editing efficiency was calculated as the ratio of white colonies on the X-Gal plate to the total number of transformants. Normalized editing efficiency was calculated as the editing efficiency × (total CFU obtained with lacZ-targeting sgRNA/total CFU obtained with control sgRNA).

Whole-genome sequencing.

Edited strains and strain H37Ra harboring the helper plasmids pNHEJ-recX or pNHEJ-recA_mu were incubated at 37°C until they reached an OD600 of 0.6 to 0.8. _M. marinum and M. tuberculosis chromosomal DNA was extracted as previously described (62) and then sonicated into fragments of less than 500 bp. The fragments were treated with End Prep enzyme mixture for end repair, 5′-end phosphorylation, and dA tailing in one reaction, followed by a T-A ligation reaction to add adaptors to both ends. Size selection of the adaptor-ligated DNA was then performed using the AxyPrep Mag PCR clean-up kit (Axygen), and fragments of approximately 410 bp (with an approximate insert size of 350 bp) were recovered. Each sample was then amplified via PCR for eight cycles using the P5 and P7 primers, both of which carry sequences that can anneal with the flow cell to perform bridge PCR. The P7 primer also carries a 6-base index that allows multiplexing. The PCR products were cleaned up using the AxyPrep Mag PCR clean-up kit (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified with a Qubit2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Sequencing was carried out using a 2 × 150-bp read length, and approximately 500-fold coverage for the genome size was expected.

Construction of the CRISPR knockout library.

A total of 88 sgRNA-expressing plasmids targeting 44 toxin genes were constructed individually. Purified pooled plasmids were transformed into M. tuberculosis H37Ra cells carrying pNHEJ-recX-sacB. The resulting transformants were first picked for analysis of the transformed sgRNAs and then for verification of the genome editing via PCR and sequencing analysis.

Data availability.

The data that support the findings of this study are available from the corresponding authors upon request. Short-read data in this study have been deposited at the NCBI Sequence Read Archive (SRA) with the accession number PRJNA559662.

Article TitleA CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing inMycobacterium tuberculosis

Abstract

New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis. Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis. Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coli.


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