The genome editing method uses a CRISPR-Cas12a system, including Cas12a and corresponding crRNA(s), to eliminate unmodified wild-type cells by double-stranded DNA cleavage, in conjunction with the phage-derived λ-Red recombinases to facilitate recombination between the chromosome and the donor DNA. With CRISPR-Cas12a-mediated restriction to eliminate unmodified cells, homologous sequences in the donor plasmid as templates, and λ-Red to accelerate recombination, we assumed to achieve genomic modifications when all conditions were met. It is worth mentioning that, differing from linear templates (PCR products or DNA oligos), circular templates (donor DNAs in high copy-number plasmids with the pUC origin) were adopted in this study to increase their concentration. We believed that the increase in template concentration should make multiplex engineering possible. The two-plasmid system was composed of a helper plasmid and a donor plasmid (Table 1). The helper plasmid series comprised the λ-Red recombinase expressed under the control of the anhydrotetracycline (aTc)-inducible promoter P_tet, and the Cas protein (Cas9 or Cas12a) expressed under the control of the arabinose-inducible promoter P_araB (Guzman et al., 1995; Cha et al., 1997) (Figure 1A). Because the P_araB promoter is repressed in presence of high glucose concentration (Guzman et al., 1995), glucose was used for inhibition of the expression of Cas12a. The donor plasmid series comprised the guide RNA(s) expressed constitutively via the J23119(SpeI) promoter (Liu et al., 2011), and donor DNA(s) as editing template(s) comprising a heterologous gene flanked by two 500 bp homologous arms, a left arm (LA) and a right arm (RA) (Figure 1B). Schematic maps of the donor plasmid p46Cpf1 with the native FnCas12a (FnCpf1) gene and the helper plasmid pTc-GLP which provides donor DNAs and crRNAs for three different sites are shown in Figures 1A,B, respectively. The plasmid construction process was described in Supplementary Tables S1, S2.

In the two-plasmid system, effects of different components on inducible cell killing were tested (Figure 2). Cas12a processes the transcript from the donor plasmid to generate mature crRNAs (Fonfara et al., 2016). Guided by the crRNA, Cas12a finds the genomic target and induces a double-strand break (Zetsche et al., 2015) (Figure 2A). Recombination occurs between the genomic target and the donor DNA mediated by λ-Red (Sharan et al., 2009). We introduced intact or deficient donor plasmids into bacteria harboring the helper plasmid p46Cpf1 by electroporation. The total colony number of forming units (CFUs) refers to the number of competent cells prepared for each electroporation. The rate of CFU to total CFU indicated the survival chance of cells with intact or deficient two-plasmid systems. It was shown that Cas12a and crRNA were necessary for inducible cell killing (Figure 2B, cases a, b, and e). Furthermore, donor DNA has an important impact on recovery from the double-strand break caused by the CRISPR-Cas12a as the editing template (Figure 2B, cases c and e). Although λ-Red was considered to accelerate recombination, more cells recovered from cleavage by Cas12a when λ-Red was not induced (Figure 2B, cases d and e), which was probably due to a reduction of metabolic stress. It was confirmed that the inhibition of Cas12a expression by the addition of glucose prevented cell death caused by the double-strand break. This phenomenon guaranteed the coexistence of the helper plasmid with the Cas12a gene and the donor plasmid with the crRNA. After the necessity of the different components of the two-plasmid system was confirmed, several parameters were adjusted to achieve a high integration efficiency. Also, the λ-Red requirement was evaluated in the next section.

We proposed to introduce the two plasmids into E. coli MG1655 sequentially, and incubate the obtained colonies without induction. After a certain number of cells had amplified, λ-Red and Cas12a were induced to accelerate recombination and selection. Cultures were spread on plates with L-arabinose to induce the expression of Cas12a consistently. We selected 12 colonies from each plate for PCR genotyping (Supplementary Figure S1). The integration efficiency was defined as the rate of the number of modified colonies to the total number of colonies that had been genotyped. A general outline of this method is shown in Figure 1C.

We tested the recombination system by introducing a gene at a single locus in the E. coli genome. To avoid affecting the growth of the modified cells, five sites with non-essential genes (araD, galK, lacZ, mutS, and pyrF) were selected for the integration of heterologous genes, and crRNAs targeting these genomic sites (Supplementary Table S3) were designed.

Firstly, it was reported that the assistance of λ-Red was necessary in the genome editing technologies based on CRISPR-Cas9 (Jiang et al., 2015; Pyne et al., 2015; Bassalo et al., 2016), and the λ-Red requirement was also evaluated for this strategy. In the helper plasmid, the λ-Red proteins are expressed under the control of an aTc-inducible promoter, P_tet. Therefore, it was possible to regulate λ-Red production using the inducing agent – aTc. The helper plasmid p46Cpf1 and the donor plasmid pTc-P were used to edit the pyrF locus in both the induced and non-induced backgrounds. While the two-plasmid system worked in the induced backgrounds at two different concentrations, almost no modified cells were observed in the non-induced backgrounds (Figure 3A), underlining the necessity of λ-Red for efficient recombination. Although, from the experimental results, an increase of the aTc concentration from 40 to 80 ng/μl did not have a significant effect on integration efficiency or the amount of surviving cells, we chose the aTc concentration of 80 ng/μl in subsequent experiments to ensure the adequate supply of λ-Red recombinases.

Secondly, we aimed to improve the expression of Cas12a for effective selection. One option was to use a strong promoter or ribosome binding site (RBS) to augment the amount of messenger RNA (mRNA). The Cas12a gene, however, is very large (∼4 kb), which causes a great burden for the cells when it is transcribed at high levels. Thus, codon optimization of the Cas12a gene was conducted to improve the production of mature Cas12a while keeping the mRNA content at the original level. To this end, two different codon optimization strategies were employed. The Cas12a (Cpf1) gene in p46Cpf1-OP1 was optimized using OPTIMIZER (Puigbo et al., 2007), whereas in p46Cpf1-OP2, another tool, JCat (Grote et al., 2005) was utilized (Supplementary Table S3). The same donor plasmid, Tc-P (Table 1), was harnessed for recombination at the pyrF site, while three different donor plasmids were tested that supplied the λ-Red proteins and Cas12a in different quantities. The results are shown in Figure 3B and Supplementary Figure S2. Although the results were not statistically significant to prove a more efficient recombination or selection assisted by p46Cpf1-OP1 or p46Cpf1-OP2, an increased average integration efficiency was observed after the Cas12 gene was codon optimized. We presumed that production enhancement of mature Cas12a had a positive impact on the recombination assays.

Thirdly, we tested different lengths of the homology arms. At the pyrF site, recombination templates with 50 bp (pTc-P-50bp), 100 bp (pTc-P-100bp) and 500 bp (pTc-P) homology arms were used. As a result, 50 bp homology arms were insufficient for recombination, and integration efficiency increased dramatically when the homology arms were elongated from 100 bp to 500 bp (Figure 3C).

Finally, the effect of different crRNAs was explored. We designed two crRNAs at each site for araD and galK, respectively. The plasmids pTc-G and pTc-A expressed crRNAs targeting the leading strand in the E. coli genome, while those expressed by pTc-G2 and pTc-A2 targeted the lagging strand. It was interesting that the two crRNAs targeting the araD site had markedly different appearance. At the galK site, however, the results of using different crRNAs were similar (Figure 3D). There was not enough evidence to draw a conclusion on effects of crRNAs targeting different strands. Nevertheless, it is inferred better to design at least two crRNAs for a locus in order to ensure a functional one.

The 5 donor plasmids (Tc-A2, Tc-G, Tc-L, Tc-M, and Tc-P), providing a series of paired crRNAs and donor DNAs, were combined in the recombination assays with the helper plasmid p46Cpf1-OP2. In each round of experiment, 12 colonies were genotyped by PCR, and one of the resulting modified clones was verified by DNA sequencing to confirm the integration of the heterologous gene (Supplementary Figure S3). As shown in Figure 4, the integration efficiency was almost 100% at each site. The five loci (araD, galK, lacZ, mutS, and pyrF) are scattered in different places on the chromosome, indicating that the developed method is applicable on the whole genome.

We next investigated if this method can be used to simultaneously insert genes at multiple loci of the E. coli chromosome.

Firstly, we attempted to insert genes at two sites simultaneously. Different combination of two cRNAs against the three selected target sites (galK, lacZ, and pyrF) were tested. As shown in Figure 5A, the introduction of both heterologous genes was confirmed in more than 40% of the colonies when p46Cpf1-OP2 was utilized as the helper plasmid.

Subsequently, simultaneous insertion in all the three loci was conducted using the donor plasmid pTc-GLP and the helper plasmid p46Cpf1-OP2 (Figure 5A and Supplementary Figure S4). As we had anticipated, the integration efficiency decreased dramatically with the increase in the number of simultaneous insertions. Nevertheless, simultaneous gene insertion into three sites was achieved with an acceptable efficiency. One colony with all three desired modifications appeared on average among every five colonies, meaning that researchers should be able to obtain the desired strain through limited work spent on PCR genotyping.

Furthermore, several crRNAs can be arranged in a CRISPR array that can be processed by Cas12a itself (Fonfara et al., 2016). This feature of the CRISPR-Cas12a system makes it convenient for the expression of crRNAs targeting multiple loci. In order to compare different ways of supplying crRNAs, pTc-arrayLP and pTc-arrayPL were constructed, with crRNAs expressed in a CRISPR array in different orders. By contrast, in pTc-PL the crRNAs were expressed separately. Recombination assays were conducted using these donor plasmids and the helper plasmid p46Cpf1-OP2.Importantly, supplying crRNAs in a CRISPR array did not reduce the integration efficiency (Figure 5B).

To explore the differences of the efficiency of Cas12a and Cas9 in recombination and counterselection, both CRISPR-Cas systems were employed to build helper plasmids and donor plasmids. Recombination at a single locus, two loci and three loci was tested. As shown in Figure 6, the systems based on Cas9 and codon-optimized Cas12a had similar performance.

Assisted by CRISPR-Cas12a or CRISPR-Cas9, the integration efficiency at a single locus was almost 100% (Figures 4, 6). However, at multiple separate loci, it might decreased significantly. For example, at the galK and pyrF loci, the integration efficiency was about 40% (Figure 5A) and it dropped to about 20% when performing recombination simultaneously at 3 loci (Figure 6). It was reported that cells escaping from Cas9 cleavage might carry a defective CRISPR system (Cui and Bikard, 2016). The similar performance of CRISPR-Cas12a and CRISPR-Cas9 in the two-plasmid system convinced us that cells could also escape from Cas12a cleavage due to the deficiency in the CRISPR system. When double-strand breaks were introduced to multiple separate loci in the chromosome at the same time, it might be difficult to repair all the breaks through homologous recombination. Less cells survived through recombination between the chromosome and the donor DNA, while more cells carried a defective CRISPR system, leading to the dramatic decrease in integration efficiency.

To demonstrate the practical application of this gene-insertion method, we constructed recombinant E. coli strains for the production of ALA, an industrially useful chemical. In the past, we constructed a series of strains for the production of ALA which carry the codon-optimized mitochondrial 5-aminolevulinic acid synthase (EC: 2.3.1.37, hem1) gene on plasmids (Li et al., 2016). Although expressing genes from high-copy-number plasmids enables high output, episomal plasmids are sometimes unstable (Ganusov and Brilkov, 2002) and require the addition of antibiotics during fermentation (Godwin and Slater, 1979), which makes chromosomal integration of metabolic pathways a promising alternative (Englaender et al., 2017). In this study, we achieved to integrate the hem1 gene into the E. coli chromosome using the developed gene inserting method.

To obtain a high-yielding recombinant strain, we selected two promoters to control the hem1 gene: the constitutive 103 promoter (P₁₀₃) and the T7 promoter (P_T7). The p103-hem1 cassette (∼1.8 kb, Supplementary Table S5) was integrated into the torS site to construct the strain MG1655AX01 (Table 1 and Figure 7A). The strain MG1655AX02 (Table 1 and Figure 7A) was obtained by simultaneously integrating the T7 RNAP cassette (∼2.6 kb, Supplementary Table S5) into the lacZ site and the pT7-hem1 cassette (∼2.0 kb, Supplementary Table S5) into the torS site. In MG1655AX02, T7 RNAP was under control of the native lac promoter (P_lac), which is isopropyl-β-D-thiogalactoside (IPTG)-inducible. We introduced the plasmid pLTT05 into MG1655 to obtain the strain MG1655AX03 (Table 1), in which the hem1 gene was controlled by a T7lac promotor and T7 RNAP was expressed constitutively. These strains were used for fermentation. The best average yield of the recombinant strain MG1655AX02 was 1.55 ± 0.29 g/L, a performance better than that of MG1655AX03 with an episomal hem1 gene (Figure 7B), which was probably the result of the usage of a strong RBS. Intriguingly, MG1655AX02 had similar performance in induced and non-induced conditions while MG1655AX03 produced much less ALA when induced by IPTG. These results implied that the proteins leaking from the IPTG-inducible promoters (P_lac or P_T7lac) might be enough for efficient production of ALA and that excessive expression of T7 RNA polymerase or ALA synthase was a burden for bacteria. Although we made sure that nearby promoters in the upstream of hem1 or T7RNAP would not influence the protein expression by positioning them in the opposite direction, it was noteworthy that random sequences could somehow serve as active promoters. Thus, terminators should be added in the upstream of these genes to insulate them from outside influence.

Recently, a method based on CRISPR-Cas9 for editing the genome of the extremophilic Halomonas spp. was reported (Qin et al., 2018). Notwithstanding, CRISPR-Cas12a can be used when there are no appropriate targets for Cas9, because they recognize different PAM regions. To test the potential of CRISPR-Cas12a in editing the genome of different bacterial species, we built another two-plasmid system for genome editing in H. bluephagenesis, comprising the helper plasmid pTD-Cas12a and a donor plasmid, for example, pTtd-prpC (Table 1). Similar strategies as reported by Qin et al. (2018) were exploited to perform the deletion of prpC in the chromosome of H. bluephagenesis. pTtd-prpC was used to delete the prpC gene, and pTtd-prpC-PM was used to introduce a point mutation into the PAM sequence in the target of crRNA-prpC, generating a premature stop codon (Supplementary Figure S5). After genotyping of the resulting colonies (Supplementary Figure S5), modified cells were sequenced to confirm the mutations. Thus, it was demonstrated that the CRISPR-Cas12a system can be used for genome editing in a non-model species of bacteria, the extremophilic H. bluephagenesis.

All strains and plasmids used in this study are listed in Table 1 and Supplementary Table S1. E. coli strain Trans1-T1 (TransGen Biotech Co., Ltd, China) was used as the host strain for plasmid construction, and E. coli strain MG1655 (CGSC 6300) was used as targets for genome engineering. Primers used for plasmid construction are listed in Supplementary Table S2. Sequences encoding the synthetic guide RNA (sgRNA) (Jinek et al., 2012), crRNAs, the spCas9 gene, the wild-type FnCas12a gene (Zetsche et al., 2015) and the two FnCas12a gene variants codon-optimized were synthesized by Qinglan Biotech (Suzhou, China) (Supplementary Table S3). The selected targets for the CRISPR-Cas system in the E. coli genome are listed in Supplementary Table S3. After DNA amplification, restriction enzyme digestion, ligation and other standard molecular cloning standard procedures were performed to construct the plasmids. We used DNA purification and plasmid isolation kits from Biomed (Beijing, China). The Q5 polymerase was purchased from New England Biolabs (Beijing, China). The restriction enzymes and ligation kits were purchased from Thermo Fisher Scientific (China).

MG1655 harboring p46Cpf1 was incubated with the corresponding inducer/inhibitor (aTc and L-arabinose/glucose) and then processed into competent cells (Sharan et al., 2009). We spread 50 μL of electrocompetent cells on LB plates to calculate total CFU. Next, 100 ng of different plasmids was introduced into 50 μL of electrocompetent cells. After 1-h incubation at 30°C and 200 rpm, the cells were spread on LB plates with chloramphenicol, kanamycin and L-arabinose/glucose. CFUs were calculated after 1-day incubation at 30°C. The ratio of transformed CFU to total CFU is shown in Figure 2B. In case a, Cas12a (Cpf1) was not induced due to absence of L-arabinose and presence of glucose; in case b, c, and e, both Cas12a and λ-Red were induced due to presence of aTc and L-arabinose; in case d, λ-Red was not induced due to absence of aTc. In case a, d, and e, the helper plasmid pTc-P was used to provide the crRNA and the donor DNA; in case b, the helper plasmid pTs-P was used to provide the donor DNA while the crRNA in CRISP-Cas12a system was absent; in case c, the plasmid pcrRNA-P was used to provide the crRNA in CRISPR-Cpf1 system while the donor DNA was absent.

A general outline of this method is shown in Figure 1C.

Day 1: Grow MG1655 in LB at 37°C overnight. Next morning, add 0.2 mL of the culture to 20 ml of LB in a 100-mL shake flask and grow at 37°C to an OD₆₀₀ of 0.6–0.8. Centrifuge the culture at 4,000 g for 5 min at 4°C. Wash the cell pellet with 20 mL of ice-cold water once and then resuspended in 1 mL of ice cold water and transfer to a 1.5-mL tube. Centrifuge at 4,000 g for 2 min at 4°C. Wash the cells two more times with 1 mL of ice-cold 10% (v/v) glycerol. Resuspend the cell pellet in ice-cold 10% (v/v) glycerol in a final volume of 1 mL. Mix about 100 ng of the helper plasmid with 50 μL of electrocompetent cells then transfer into a 2-mm Gene Pulser cuvette (Bio-Rad, United States). Introduce the helper plasmid into the cells by electroporation at 1.8 kV. After electroporation, add 1 ml of LB and transfer the cells to a 1.5-mL tube. Incubate the cells at 30°C for 1 h. Plate 100 μL of the cell suspension onto an LB plate with 12.5 μg/mL chloramphenicol and incubate at 30°C 20–22 h. (Tips: Chemical transformation is feasible as well.)

Day 2: Pick an individual colony and grow in LB with 12.5 μg/mL chloramphenicol at 30°C and 200 rpm overnight.

Day 3: Add 0.2 mL of the culture of the intermediate strain to 20 mL of LB with 12.5 μg/mL chloramphenicol and 20 g/mL glucose (to inhibit transcription from the ParaB promoter) in a 100-mL shake flask and grow at 30°C to an OD₆₀₀ of 0.6–0.8. Process the intermediate strain into competent cells (same procedure as above). Introduce about 100 ng of the donor plasmid into the cells via electroporation in a 2-mm Gene Pulser cuvette (Bio-Rad) at 1.8 kV, after which add 1 mL LB liquid medium with 20 mg/mL glucose to the electroporated cells. Regenerate at 30°C for 1 h and plate 100 μL of the cell suspension onto an LB plate with 12.5 μg/mL chloramphenicol, 50 μg/mL kanamycin and 20 mg/mL glucose and incubate at 30°C for 20–22 h. (Tips: Chemical transformation is feasible as well.)

Day 4: Pick an individual colony and grow in LB with 12.5 μg/mL chloramphenicol, 50 μg/mL kanamycin and 20 mg/mL glucose at 30°C and 200 rpm overnight.

Day 5: Add 10 μL of the resulting overnight seed culture to a culture tube containing 1 mL LB medium with 50 μg/mL kanamycin and 12.5 μg/mL chloramphenicol. Cultivate at 30°C and 200 rpm for 6 h, then add 5 mg/mL L-arabinose and 80 ng/mL aTc to the mixture. After a further 2 h of incubation, spread a serial dilution of the harvested culture (100, 10, and 1 μL as well as 0.1 μL of the culture) on LB plates supplemented with 5 mg/mL L-arabinose, 50 μg/mL kanamycin and 12.5 μg/mL chloramphenicol. (Tips: It is recommended that inducers be added when the turbidity of the culture is visible to the naked eye. In case the bacteria grow slowly, incubate the culture over night before spreading it on plates.)

Day 6: After overnight incubation at 30°C, confirm the corresponding strains by PCR genotyping.

PCR genotyping was carried out to distinguish wild-type cells from those with the desired modifications. The corresponding primers are listed in Supplementary Table S4. Hotstart Taq polymerase, which does not possess the 3^′ to 5^′ exonuclease activity was purchased from TransGen Biotech Co., Ltd., China. Gene insertions were confirmed by PCR amplification using a forward primer targeting a sequence upstream of the genomic locus (at a distance of more than 500 bp from the transcription-initiation site) and a reverse primer binding the inserted gene. In order to detect wild-type cells, one primer of the pair was designed to bind the genomic sequence which should disappear after recombination. Twelve colonies were genotyped in each round of experiment. The schematic diagrams were provided in Supplementary Figure S1. In some cases, both genotypes were detected in a single colony (Supplementary Figures S2B,C, arrows), which means that such a colony was composed of both wild-type and modified cells. A mutant colony was confirmed as successfully modified only if no wild-type cells were detected. The integration efficiency stands for the ratio of successfully modified colony number to total colony number in the PCR genotyping test.

The confirmed colonies were picked and grown in 2 mL of LB at 42°C and 200 rpm overnight. Next morning, the culture was diluted and spread onto LB plates. After 12 h’ incubation at 37°C, 24 resulting colonies were picked and each of them was inoculated onto three distinct plates – an LB plate supplemented with 50 μg/mL kanamycin, an LB plate supplemented with 12.5 μg/mL chloramphenicol and a further LB plate without antibiotics. The colonies sensitive to both antibiotics were confirmed as plasmid-free.

The plasmids p46Cpf1-OP2 and pTc-torS-p103-hem1 were used to construct the recombinant E. coli MG1655AX01 with the hem1 gene controlled by P₁₀₃ inserted into the torS site. The plasmids p46Cpf1-OP2 and pTc-lacZ-T7RNAP-torS-pT7-hem1 were used to construct the recombinant E. coli MG1655AX02 with the hem1 gene controlled by P_T7 inserted into the torS site and the T7 RNA polymerase gene into the lacZ site. At the torS site, the primer pair AX069/AX070 (Supplementary Table S4) was used in genotyping PCR to verify the modified cells, while the primer pair AX067/AX068 (Supplementary Table S4) was used to verify wild-type cells.

The tested strains were incubated in 20 mL of LB medium at 37°C and 200 rpm overnight to form seed cultures. 1 mL of the seed culture was inoculated into a 50-mL shake flask containing 20 mL of pH-adjusted LB medium (10 g/L tryptone, 5 g/L yeast extract, 83.4 mM K₂HPO₄∙3H₂O, 216.6 mM KH₂PO₄, 3.0 g/L glycine and 6.0 g/L succinic acid). Where appropriate, 2 mM isopropyl-β-D-thiogalactoside (IPTG) was added at the beginning of the fermentation. The fermentation was conducted at 30°C and 200 rpm. After 18 h of incubation, an additional 10.0 g/L glucose, as well as 5.0 g/L glycine and 10.0 g/L succinic acid were added. The supernatant containing ALA was harvested after 48 h of fermentation. The experiments were carried out in triplicate and the ALA produced by the culture was quantified using a classical method (Mauzerall and Granick, 1956).

The procedures for genome editing of H. bluephagenesis using the method based on CRISPR-Cas12a were developed according to that based on CRIPSR-Cas9 (Qin et al., 2018). Generally, the helper plasmid pTD-Cas12a was introduced into H. bluephagenesis TD01 by conjugation, followed by the donor plasmid pTtd-prpC or pTtd-prpC-PM (Table 1). After at least 36 h of incubation, the resulting colonies were genotyped by PCR.