CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

da | Giu 2, 2018 | Biologia Molecolare

Corynebacterium Glutamicum, an important organism for industrial application, since it is used to produce amino acids, polymer subunits and biofuels[1]. However, editing by CRISPR/Cas9 system in order to engineer this bacteria has revealed difficult. CRISPR system is a great tool that completely changed molecular biology, needs to be developed also in C. glutamicum, to replace the classic allele exchange techniques[2]. In a recent article, Jang and collaborators described a genome editing technique Thus substituting Cas9 with the single stranded cutting enzyme Cpf1. Difficulties have been taken to account using the well-known Cas9 method. In fact, the problem of using CRISPR/Cas9 in C. glutamicum might be due to Cas9 toxicity in this bacterium as previously reported in Sara Cleto et al. work[3]. Jang and collaborators now attemed, for the first time, to use the CRISPR-Cpf1 in C. glutamicum, optimized the conditions and applied their results to improve L-proline production in this bacteria, an important amino acid in industrial field.

They constructed three plasmids harbouring Cas9 gene under the control of different promoters, none of these generate transformants of Corynebacterium glutamicum (ATCC13032), demonstrating toxicity of Cas9. As a consequence, another system was chosen: Cpf1 nuclease of Francisella novicida. Using a plasmid carrying Cpf1 gene, they could obtain high transformation efficiency, almost equalizing those of the control, indicating a good surviving index. Then, two different modalities of transformation were elaborated, to optimize CRISPR-Cpf1 technique. The first characterized by the usage of a all-in-one plasmid pXMJ19ts carrying FnCpf1 and the crRNA, a RNA that targets the nuclease to the locus of interest, for crtYF. The second by the use of plasmids added at different times.

The first one carrying Cpf1 gene (in red in the figure), the second harbouring the sequence for crRNA (in yellow in the figure). In both cases, transformation efficiencies were relevant but lower than those of controls, a fundamental prerequisite for a positive control. At this stage, a specific homologous recombination is researched. The two-plasmids system is used, implemented with RecT, a protein that facilitates the homologous recombination (marked in red in the figure). Besides, oligonucleotides homologous to the target sequence but presenting a little designed difference, highlighted in green in the figure, were added. Cleavages on crtYF targeted by crRNA and obtained by Cpf1 nuclease cause a damage resolved by homologous recombination, that takes place thanks to oligonucleotides. At the end, the sequence presents a modification of 2 pb (from GC to TT) induced by the oligonucleotides, creating a specific HpaI restriction site (TTAAC) into the crtYF locus. Restriction digestion and PCR amplification, followed by Southern blot indicates whether the modification has taken place. Recombination efficiency is much higher if the oligonuceltodies target the lagging strand instead of the leading strand, as confirmed by Jan-Peter van Pijkeren et al.[4], even if there is not an explanation of this phenomenon. Different plasmids (but always following the two-plasmids strategy) were then prepared, to investigate other kind of modifications. The results are not impressive: deletions of 50 pb and 500 pb showed an editing efficiency of 15% and 0% respectively. The percentage increased (40%) if the sequence to delete was of 17 pb. At the end using a different target (argR), the excellent editing efficiency of 100% by this tool for 2 pb substitutions.

This modification was also tested in other Corynebacterium species of industrial interest and the results were again a 100% of editing efficiency. Next, the all-in-one plasmid strategy has been tested. At first deletions at crtYF locus were scanned: for sequence of 705 pb editing efficenvy was 15%, it was 10% for 7.5 kb length. Then, insertion of a gene was detected. The gene was tdcb, flanked by homologous arms into the plasmid, which combine with specific regions into the genome, where the gene will be inserted. Editing efficiency in this case was only 5%. A mutation (R1218A), detected by BLAST[5], induce nickasic activity by Cpf1 (FnCpf1R1218A). However, the number of surviving transformants did not decrease in this case: a positive selection is not possible.

C. glutamicum is mostly used in industrial application, so the authors decided to test the successful modification of 2 pb obtained in the work to increase the L-proline production by this strand. L-proline is an amino acid very useful for pharmaceutical and medical applications, typically produced by C. glutamicum. L-proline can inhibit γ-glutamil kinase by a negative feedback, blocking the first step of the pathway for its production. Modifying the ProB gene, that encodes for γ-glutamil kinase enzyme, L-proline production is supposed to increase. At first, using BLAST, comparing ProB sequences from E. coli, B. Thailandensis and C. glutamicum, the allosteric site on which L-proline acts is detected, in particular the G149 amino acid. The following step was to construct plasmids carrying the crRNA for ProB gene. These are enriched with PAM sequences found flanking the G149 into C. glutamicum genome, in order to avoid off-target matches by crRNA and other plasmids were enriched respectively with PAM1, PAM2 and PAM3, allowing a better recognition of the target sequence by crRNA, as confirmed in literature[6]. Then, oligonucteotides were designed in order to present a modification at G149 amino acid, as described in the figure. In addition, silent mutations were induced on PAM sequences flanking G149, to avoid dangerous cleavages on the oligonucleotides. After different attempts, twenty olgionucleotides were prepared, all showing good editing efficiency, up to 90%. C. glutamicum are transformated with the plasmids PJYS1 and PJYS2-ProB series, besides, oligonucletoides desinged are added, as can be appreciate by part A and B of the figure. At the end, to test the expected L-proline incremented production, fermentations take place. Volumes are small, 600 μL. By HPLC the amount of L-proline produced for each fermentation is misured. This data is combined with sequencing of the ProB gene recombined, in order to match the presence of a new amino acid on the sequence to a certain production of L-proline. From the results summarized in the picture, the G149K substitutions, with lysine instead of glycine, is the best, since it induces a seven-times improved production of L-proline (6.6 g/L in 72 hours).

In order to resume goals reached and those not yet, it’s fair to start mentioning the failure of Cas9 and the successful application of Cpf1. In particular, this article demonstrates the ability of authors in avoiding one method well-known as the Cas9 system, finding out a solution with another technique less-known as using the Cpf1 system. The most interesting aspect that needs to be investigated is the toxicity of Cas9 for C. glutamicum: BLAST revealing the absence of possible off-target cleavages is a good starting point, but much more has to be done. The hypothesis that the toxicity is due to a too strong binding of Cas9 nuclease with the genome is confirmed by other studies[3], but not yet described. In addition, it shouldn’t be forgotten that Cas9 and Cpf1 nucleases have really different dimensions and cleavages properties, these characteristics could explain the opposite results taken in account in this work. Secondary, Yu Jiang et al. manage to optimize a plasmids-mediated technique that requests reduced time of work and allows the Cpf1 system to perform very efficiently, in particular for 2 pb substitutions. 100% of editing efficiency is an excellent result, but for all the other modifications researched the percentages are really too low and they need to be improved. For sure, the 2 pb substitution has to be exploited, also for different industrial applications. However, substitutions of sequences longer than 2 pb should be studied, as deletions of short sequences. Finally, the authors are cleaver in the choice of the application for this editing technique: as results attesting the enhanced production of L-proline confirm. However, scale-up experiments should be performed, in order to evaluate if the modification of ProB gene described in this work is really applicable in industrial field. In fact, volumes of fermentation was too small (less than 1 mL) to predict the goodness of this edited C. glutamicum strand for the production of L-proline.

Figure. (A) C.glutamicum wild type is able to produce < 1 g/L of L-proline. First transformation is shown: PJYS1 series plasmid is inserted, it harbours genes for recT and FnCpf1 (in red). (B) Second transformation: the PJYS2 plasmid carrying the crRNA (in yellow) to target the nuclease on ProB (in blue) is added. PAM sequences, present in ProB gene and flanking the G149 target site, enrich the crRNA for a more precise recognition by the nuclease. Besides, oligonucleotides for homologous recombination are added. They are modified (green segment) in order to induce a modification of G149, plus, they carry also silent mutations on nucleotides flanking the target site (PAM), in order to avoid undesired recognition by Cpf1 nuclease. (C) C. glutamicum has been modified on ProB gene, glycine in 149 is substituted by lysine (in green, this is the best case). As a consequence, L-proline production is attested at 6.6 g/L (seven-times improved, comparing with the wild type).

References

  1. Wendisch, V. F., Jorge, J. M., Perez-Garcia, F. & Sgobba, E. Updates on industrial production of amino acids using Corynebacterium glutamicum.  J. Microbiol. Biotechnol.32, 105 (2016).
  2. Tan, Y., Xu, D., Li, Y. & Wang, X. Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum. Plasmid67, 44–52 (2012).
  3. Cleto S, Jensen JV, Wendisch VF, Lu TK. Corynebacterium glutamicummetabolic engineering with CRISPR interference (CRISPRi) ACS Synth Biol. 2016;5:375–385.
  4. van Pijkeren J.P., Neoh K.M., Sirias D., Findley A.S., Britton R.A. Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri, Bioengineered, 2012, vol. 3 (pg. 209-217)
  5. Yamano, T. et al.Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA. Cell 165, 949–962 (2016).
  6. Leenay, R. T. et al.Identifying and visualizing functional PAM Diversity across CRISPR-Cas Systems.  Cell. 62, 137–147 (2016).

Paolo Costa

Master Industrial Biotechnology student

Lorenzo Pacifici

Master Industrial Biotechnology student

 

Apri un sito e guadagna con Altervista - Disclaimer - Segnala abuso - Privacy Policy - Personalizza tracciamento pubblicitario