Materials and Methods

CRISPR-Cas12a-Assisted Recombineering in Bacteria

MATERIALS AND METHODSStrains, media, and growth conditions. E. coli MG1655, Y. pestis KIM6+, and M. smegmatis mc2155 were used in this study. E. coli and Y. pestis were grown in LB medium supplemented with appropriate antibiotics (25 μg/ml kanamycin, 100 μg/ml ampicillin, or 30 μg/ml chloramphenicol). M. smegmatis was grown in Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween 80 and 0.2% glycerol or on Middlebrook 7H10 agar supplemented with the appropriate antibiotic (25 μg/ml kanamycin, 50 μg/ml hygromycin, or 50 μg/ml zeocin). Appropriate concentrations of anhydrotetracycline (ATc) were added to the M. smegmatis cultures when necessary. To facilitate the screening of recombinants in M. smegmatis, a GFP reporter gene was inserted into the Ms5635–Ms5634 locus of the chromosome using recombineering (52).Plasmids. The FnCpf1 open reading frame (ORF) sequence was cloned into pKD46 (13) using Gibson cloning to yield pKD46-Cas12a series plasmids (Fig. S1A). The pKD46-Cas12a series plasmids, which contain a temperature-sensitive replicon, can be cured at 42°C. The pre-crRNA cassette containing the gfp gene was commercially synthesized and cloned together with the sacB gene into the modified pACYC184 vector to yield pAC-crRNA series plasmids (Fig. S1). The gfp gene, which is flanked by BpmI and BsaI restriction enzyme sites, was used as a selection marker for protospacer cloning (Fig. S1). Two complementary oligonucleotides containing the target sequence adjacent to 5′-YTN-3′ were synthesized, annealed to yield a protospacer cassette with BpmI or BsaI overhangs at the 5′ and 3′ ends, respectively, and then cloned into the pAC-crRNA plasmid (Fig. S1). The research presented in this study was conducted using the pKD46-Cas12a-Amp and pAC-crRNA-Cm plasmids. pKD46-Cas12a-Cm and the other pAC-crRNA series plasmids were also tested and yielded results similar to those obtained using npKD46-Cas12a-Amp and pAC-crRNA-Cm plasmids (data not shown).A codon-optimized FnCpf1 ORF sequence (optimized with JCat) (53) (Fig. S2) under the control of the Pmyc1tetO promoter was commercially synthesized (GENEWIZ) and cloned into pMV261 and pJV53 to yield pMV261-Cpf1 and pJV53-Cpf1, respectively (Fig. S4A). The pre-crRNA cassette was commercially synthesized and cloned into pSL003 (54) to yield pCR-Zeo (Fig. S4B). The hygromycin resistance gene was amplified by PCR from pSL002 and cloned into pCR-Zeo to replace the zeocin resistance gene, yielding pCR-Hyg (Fig. S4B). The Cas12a gene was amplified by PCR from pMV261-Cpf1 and then ligated into pcrRNA-ctrl and pcrRNA-gfp1 digested with KpnI and NotI, to yield pCpf1-ctrl and pCpf1-gfp, respectively. To mutate a particular gene in mycobacteria, two complementary oligonucleotides containing the target sequence adjacent to ′-YTN-3′ were synthesized, annealed to yield a protospacer cassette with BpmI and HindIII overhangs at the 5′ and 3′ ends, respectively, and then cloned into pCR-Zeo or pCR-Hyg. All plasmids constructed in this study are listed in Table S1. The oligonucleotides used in this study are listed in Table S2.M. smegmatis growth assay. M. smegmatis cells harboring pMV261 or pMV261-Cpf1 were inoculated into 3 ml of 7H9 broth supplemented with kanamycin and grown overnight with shaking at 37°C. The overnight cultures were diluted to an optical density at 600 nm (OD600) of 0.02 in 50 ml of 7H9 broth supplemented with kanamycin and appropriate concentrations of ATc. These cultures were grown overnight with shaking at 37°C, and samples were taken at appropriate time points to determine the OD600.Plasmid interference assay. The plasmid interference assay was carried out as previously reported, with minor modifications (37). Briefly, M. smegmatis mc2155 harboring pCpf1-ctrl or pCpf1-gfp was transformed with pJV53 or pJV53-GFP, respectively. The transformants were plated onto 7H10 medium supplemented with or without 50 ng/ml ATc and then grown for 3 days at 37°C. The colonies were counted to calculate the CFU.Preparation of recombinogenic DNA. For the ssDNA oligonucleotide recombineering experiments, the recombinogenic oligonucleotides were synthesized, and mutations were introduced into the middle of the oligonucleotide sequences with at least 25 nt of sequence identity on both sides of the mutation site. The leading and lagging strands of the bacterial chromosomes were determined using cumulative skew diagrams (55).For the dsDNA recombineering experiments, the gfp gene was amplified from pAcGFP1 vector using primers with 45-nt homology regions of aroA to generate recombinogenic dsDNA products for aroA replacement in E. coli. To generate dsDNA homologous arms for the deletion of 2, 392, or 1,000 bp from the gfp gene in M. smegmatis, the Ms5635–Ms5634::gfp cassette with flanking regions was amplified by PCR and inserted into pUC19 to yield the plasmid pYC847. Then, pYC847 was used as the template for inverse PCR with appropriate primer sets to generate plasmids containing dsDNA homologous arms. To generate dsDNA homologous arms for the 4,000-bp deletion, a 539-bp DNA fragment downstream of Ms5634 and a 596-bp DNA fragment upstream of Ms5635 were amplified by PCR and cloned into pUC19 to yield pYC848. These plasmids were digested with KpnI and SphI, and the digested fragments were gel purified. pYC710, pYC711, pYC799, and pYC738 were used as templates to generate pYC984, pYC985, pYC986, and pYC987, which contain the dsDNA homologous arms for the toxin-antitoxin systems Ms1277–Ms1278, Ms1283–Ms1284, Ms4447–Ms4448, and Ms5635–Ms5634, respectively. The dsDNA homologous arms generated from these new plasmids were digested with HindIII and KpnI. Ms5635–Ms5634, the hyg resistance gene, and their flanking regions were amplified by PCR from the chromosomes of wild-type M. smegmatis and the M. smegmatis Ms5635–Ms5634::hyg mutant, respectively, and used as recombinogenic fragments for the replacement of the gfp gene. The primers used in this study are listed in Table S2.Cas12a-assisted genome editing. Competent cells of E. coli, Y. pestis, and M. smegmatis were prepared as previously described (17, 52, 56). For ssDNA oligonucleotide recombineering, approximately 500 ng of recombinogenic or nonrecombinogenic oligonucleotides and 100 ng of the crRNA expression plasmid were mixed and electroporated into competent cells. For dsDNA gene deletion, 700 ng of gel-purified restriction-digested product or PCR product and 100 ng of the crRNA-expressing plasmid were mixed and electroporated into competent cells. For E. coli and Y. pestis, the electroporated cells were plated and grown on LB agar supplemented with appropriate antibiotics overnight at 30°C. Recombination of lacZ was assessed by examination for the formation of white colonies and then confirmed by PCR and sequencing analysis. Recombination in Y. pestis was analyzed and verified by PCR amplification and sequencing. Plasmid-free colonies were obtained by incubating the cells in LB culture medium supplemented with sucrose at 42°C. For M. smegmatis, cells were recovered after incubation in 1 ml of 7H9 broth with 10 ng/ml ATc for 4 h at 30°C at 200 rpm and then plated on 7H10 agar supplemented with the appropriate antibiotics and 50 ng/ml ATc. After growth for 4 days at 30°C, the plates contained normal-size and tiny transformant colonies. The normal colonies were counted and used to calculate the transformation and recombination efficiencies. Recombination of gfp was assessed by examining for the loss of the GFP signal, and then each time, at least 5 GFP-negative recombinants were picked for PCR and sequencing analysis, and all of the tested colonies were confirmed to be desired recombinants. Recombination involving other genes was assessed by PCR and sequencing. To cure the helper plasmids from the M. smegmatis recombinant, the right recombinant colony was picked and grown overnight at 37°C in 7H9 medium without antibiotic. The overnight culture was diluted 1:500 into 7H9 medium and grown for 24 h at 37°C. The resultant cultures were diluted, plated, and grown for 3 days on 7H10 plates and then tested for the loss of plasmids using replica streaking on the plate with or without appropriate antibiotics.

Article TitleCRISPR-Cas12a-Assisted Recombineering in Bacteria

Abstract

The next goal of this study was to establish CRISPR-Cas12a selection in mycobacteria to enable high-efficiency recombineering. To evaluate the feasibility of this goal, we cloned and expressedFnCpf1 in mycobacteria. The gene encoding Cas12a was modified to optimize expression in mycobacteria (Fig. S2). Although Cas12a is an exogenous protein, induced expression of Cas12a (up to 100 ng · ml−1anhydrotetracycline ATc) did not strongly affect the growth ofM. smegmatis(Fig. S3A). Next, we performed plasmid interference assays to testFnCpf1 activity in mycobacteria (37). In this approach, expression of a Cas endonuclease and a crRNA on a transformed plasmid results in cleavage of the target plasmid, which is reflected as a decrease in the number of transformants. For this purpose, we constructed a plasmid for coexpression of Cas12a and a crRNA targeting thegfpgene (pCpf1-gfp) or a crRNA not targeting thegfpgene (pCpf1-ctrl).M. smegmatisharboring pCpf1-gfp or pCpf1-ctrl was transformed with pJV53 (withoutgfp) or pJV53-GFP (withgfp). Expression of Cas12a and thegfp-specific crRNA significantly decreased the number of transformants with pJV53-GFP but not the number of transformants with pJV53, whereas expression of Cas12a and the nonspecific crRNA did not affect the transformation efficiency of either pJV53-GFP or pJV53 (Fig. S3B). Taken together, these results suggest thatFnCpf1 is biologically active and can mediate targeted DNA interference inM. smegmatis.


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