CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%. Large gene deletions and insertions are also obtained using an all-in-one plasmid consisting of FnCpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3N+4 or 3N+2 days. A proof-of-concept, codon saturation mutagenesis at G149 of γ-glutamyl kinase relieves L-proline inhibition using Cpf1-assisted ssDNA recombineering. Thus, CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas9 system.

Figure 1. Overview of CRISPR-Cpf1-assisted genome editing in C. glutamicum.

A flow-chart for (a) the double-plasmid-based CRISPR-Cpf1 system for small genome alterations, and (b) the all-in-one CRISPR-Cpf1 plasmid for large gene deletions and insertions. The double-plasmid-based CRISPR-Cpf1 system includes FnCpf1 and RecT expressed from pJYS1Ptac or pJYS1Peftu (Supplementary Fig. 1), respectively, and a crRNA -expressing plasmid pJYS2 series targeting different loci in the genome (indicated as crtYf in the figure). The pJYS2 series plasmid was co-transformed with donor ssDNA into recipient cells harbouring pJYS1 expressing RecT and FnCpf1. The pJYS2 series can be cured by overnight incubation in spectinomycin-free medium, followed by subculturing the next day for the next round of editing. The final strain was obtained by overnight culturing in antibiotic-free medium at 34 °C. The total editing time is 3N+4 days for N rounds of genome editing. The all-in-one CRISPR-Cpf1 plasmid pJYS3 series consists of FnCpf1 and crRNA, and the upstream and downstream homologous arms that served as the donor DNA. The pJYS3 series can be cured by overnight culturing in kanamycin-free medium at 34 °C, followed by subculturing the next day for the next round of editing. The total editing time is 3N+2 days for N rounds of genome editing. pBL1^ts: a temperature sensitive replication derived from the pBL1 replicon of C. glutamicum; pSC101: a replication origin of E. coli; recT: recombination and repair protein from E. coli; Ptac: a tac promoter; Kn^r: kanamycin resistance gene encoded by the aminoglycoside phosphotransferase gene; pMB1: a replication origin of E. coli; rep: replication origin of C. glutamicum derived from the pGA1 replicon; Sp^r: spectinomycin resistance gene; Pj23119: a synthetic constitutive expression promoter; PlacM: a modified lac constitutive expression promoter in C. glutamicum; lacIq: lac repressor of E. coli; HR1/2: upstream/downstream homologous regions, respectively; crRNA: a matured CRISPR RNA that contains 20 bp conserved sequences and 21–24 bp targeting sequences; FnCpf1: cpf1 derived from F. novicida (NC_008601).

Figure 2. Optimization of double-plasmid-based CRISPR-Cpf1-ssDNA recombineering in crtYf of C. glutamicum.

(a) Growth of C. glutamicum cells expressing the nuclease, with or without the combined expression of crRNA targeting crtYf. Asterisks indicate c.f.u. were not detected. NA, not applicable. (b) Overview of ssDNA-mediated nucleotide substitution in crtYf. (c) C. glutamicum cell growth and the mutation efficiency of crtYf after editing by 59 nt (O_crtYf59-1) or 75 bp (O_crtYf75-) template oligonucleotide targeting the lagging strand mediated by the pJYS2_crtYf and pJYS1 series carrying RecT under the control of various promoters. The mutation efficiency was determined by colony PCR, followed by HpaI digestion, as indicated in Supplementary Fig. 2. (d) C. glutamicum cell growth and the crtYf mutation efficiency after editing by a 59 bp lagging (O_crtYf59-1) or leading (O_crtYf59+) oligonucleotides mediated by the pJYS1Ptac/pJYS2_crtYf system. One of several parallel experiments calculating the mutation efficiency as determined by HpaI digestion is shown in e. (e) Sixteen transformants derived from lagging or leading strand targeting oligonucleotide (59 bp O_crtYf59-1 or O_crtYf59+) -mediated, pJYS1Ptac/pJYS2_crtYf -based CRISPR-Cpf1 recombineering experiments were screened by colony PCR, followed by HpaI digestion, to identify recombinants in the crtYf locus. A 2.9 kb fragment indicates wild-type genotype, whereas the presence of 1.6, 1.1 and 0.2 kb fragments indicate recombinant genotypes. (n/N): n, number of correctly edited, positive transformants; N, number of transformants tested. DNA ladder mix (GeneRuler, Thermo Scientific) was used as a marker. (f) Ten representative crtYf recombinants identified by HpaI digestion were further sequenced, which revealed the substitution of GC by TT for all samples, as expected. Details in transformants recording and editing efficiency calculation are listed in Supplementary Data 1. Experiments were performed in duplicates. Bar represents mean±s.d.

Figure 3. Double and all-in-one plasmid-based deletion and insertion in the crtYf locus of C. glutamicum.

(a) Overview of crtYf editing by the ssDNA-mediated double-plasmid-based CRISPR-Cpf1 system (pJYS1Ptac/pJYS2_crtYf) or the all-in-one CRISPR-Cpf1 plasmid (pJYS3 series). The editing efficiency and numbers of c.f.u. of the corresponding transformants were determined by colony PCR and calculated as indicated in Supplementary Data 1 and Supplementary Fig. 3. (b) C. glutamicum growth after transformation with CRISPR-Cpf1 plasmids (pJYS3_crtYf, pJYS3Peftu_crtYf, pJYS3Psod_crtYf, pJYS3Ptac_crtYf, pJYS3Ptrc_crtYf, or pJYS3Ptet_crtYf) containing FnCpf1 under the control of various promoters (PlacM, Peftu, Psod, Ptac, Ptrc or Ptet) and the crRNA targeting crtYf locus. CRISPR-Cpf1 plasmid pJYS3 lacking crRNA was used as a control. The in vivo activity of FnCpf1 and the predicted FnCpf1 nickase R1218A were compared. Details in transformants recording and editing efficiency calculation are listed in Supplementary Data 1. (c) CRISPR-Cpf1 plasmid-curing efficiency for iterative genome manipulations. Cells carrying double plasmids pJYS1Ptac/pJYS2_crtYf or pJYS3_ΔcrtYf were cultured overnight in liquid BHISG with or without the indicated antibiotics at the indicated temperatures. Cultures were diluted and plated on BHISG plates with or without the indicated antibiotics and incubated for 48 h at 30 °C. The relative plasmid-curing efficiency was calculated by dividing the number of c.f.u. obtained from antibiotic-containing plates by the c.f.u. obtained from plates lacking the corresponding antibiotics. Experiments were performed in duplicates. Bar represents mean±s.d.

Figure 4. CRISPR-Cpf1-assisted recombineering in Corynebacterium strains.

(a) Growth of Corynebacterium strains when transformed with pJYS1Ptac, the double CRISPR-Cpf1 plasmid pJYS1Ptac/pJYS2_crtYf, or pJYS1Ptac/pJYS2_crtYf, and the 59 bp oligonucleotide, O_crtYf59-1, targeting lagging strand. Stars indicate no colony was detected. Details in transformants recording and editing efficiency calculation are listed in Supplementary Data 1. Experiments were performed in duplicates. Bar represents mean±s.d. (b) Five transformant colonies of the indicated Corynebacterium strains derived from an O_crtYf59-1 -mediated, pJYS1Ptac/pJYS2_crtYf -based CRISPR-Cpf1 recombineering were analysed by colony PCR, followed by HpaI digestion, to identify recombinants in the crtYf locus. A 2.9 kb fragment indicates the wild-type genotype, whereas the presence of 1.6, 1.1 and 0.2 kb fragments indicate a recombinant genotype. A DNA ladder (GeneRuler) was used as a marker.

Figure 5. Application of CRISPR-Cpf1 system for codon saturation mutagenesis of proB encoding γ-glutamyl kinase.

(a) Application of pJYS1Peftu/pJYS2_proB3 double-plasmid-based CRISPR-Cpf1 system for genomic in situ site-directed codon saturation of proB coding for γ-glutamyl kinase. A mixture of 20 different oligonucleotides was used to target codon 149 in cgProB. One hundred and ninety colonies were screened by direct 96-well fermentation and high-pressure liquid chromatography (HPLC) measurement of L-proline. Codon 149 of ProB was sequenced in 42 productive mutants. (b) Sequence alignment of ProBs and binding mode of L-proline. The conserved residues of ProBs from E. coli (ecProB), B. thailandensis (btProB), and C. glutamicum (cgProB) are shaded. The backbone of cgProB is shown in a ribbon model on which several key residues are shown in a stick model. Possible hydrogen bonds are indicated by dashed lines. (c) Cell growth following transformation with the pJYS1Peftu and pJYS2 series targeting the indicated three PAM regions near codon 149 of ProB as indicated in e. Details in transformants recording and editing efficiency calculation are listed in Supplementary Data 1. Experiments were performed in duplicates. Bar represents mean±s.d. (d) Effect of multiple changes on oligo recombination frequencies. Three PAM sequences near G149 of ProB are highlighted in yellow, and the wild-type codon 149 is underlined in bold. Double site-directed mutagenesis of F152FG149D and A146AG149D, and multiple mutageneses of A146AT147TT148TG149D, A146AT147TT148TG149A, A146AT147TT148TG149C, and A146AT147TT148TG149F were performed. (e) Forty-two proB recombinants with substitutions at codon 149 (table) screened by 96-well fermentation exhibited different degrees of L-proline formation (blue bars). Experiments were performed in duplicates. Bar represents mean±s.d.