# CRISPR/anti-CRISPR genome editing in Clostridium beijerinckii

Rémi Hocq, Gwladys Chartier, Nicolas Lopes Ferreira, François Wasels

► To cite this version:

Rémi Hocq, Gwladys Chartier, Nicolas Lopes Ferreira, François Wasels. CRISPR/anti-CRISPR genome editing in Clostridium beijerinckii. Journal of Biotechnology, 2026, 409, pp.165-169. 10.1016/j.jbiotec.2025.11.002. hal-05454338

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# CRISPR/anti-CRISPR genome editing in *Clostridium beijerinckii*

Rémi Hocq<fnref n="1" />, Gwladys Chartier, Nicolas Lopes Ferreira, François Wasels*

IFP Energies nouvelles, 1 et 4 avenue de Bois-Préau, Rueil-Malmaison 92852, France

## ARTICLE INFO

**Keywords:**

Anti-CRISPR

*Clostridium*

CRISPR-Cas9

Genome editing

Cell engineering

Counter-selection marker

## ABSTRACT

The development of CRISPR technologies has revolutionized genome editing. However, in bacteria, CRISPR-based methods can be difficult to implement due to the cytotoxicity of CRISPR-associated proteins, which often impair or entirely prevent transformation. In this work, we combine inducible expression of classical CRISPR-Cas9 components with the anti-CRISPR protein AcrIIA4 from *Listeria monocytogenes* to tightly regulate Cas9 activity. Using this approach, we demonstrate efficient and iterative genome editing in the genetically recalcitrant *Clostridium beijerinckii* DSM 6423. While deletion of *upp* alone was not sufficient to render the strain sensitive to 5-fluorouracil, the additional deletion of a second gene involved in the uracil salvage pathway conferred resistance to the drug and validated our gene editing strategy. Collectively, our results show that CRISPR/anti-CRISPR systems can overcome a key limitation of CRISPR-based genome editing and may offer a broadly applicable strategy for engineering otherwise intractable bacterial species.

Solventogenic *Clostridia* are industrial workhorses which have been used for more than a century in the so-called Acetone-Butanol-Ethanol (ABE) fermentation (Moon et al., 2016). With growing concerns about the sustainability of a fossil-based economy, bioprocesses converting renewable feedstocks into chemicals and fuels - such as the ABE fermentation - have gained considerable interest (Jones et al., 2023).

*Clostridium beijerinckii* and *Clostridium acetobutylicum* are the main model species used in ABE fermentation research, which with the advent of modern genetics has significantly shifted in recent years to include metabolic engineering. Rationally optimizing ABE microbes (e.g., by changing product scope and/or specificity, yield etc.) requires fast, reliable, and convenient genetic tools, which naturally led the community to develop CRISPR systems (McAllister and Sorg, 2019). These systems all rely on the selection of homologous recombination (HR) events by targeted cleavage of genomic DNA (gDNA), which cause cell death in the lack of HR-based repair of the introduced double strand break (DSB) and alteration of the target sequence. Because of the inherently low HR efficiency of *Clostridia* (Pyne et al., 2014), genetic engineering efforts are however significantly impaired by the lethality of CRISPR systems, i.e., cells being eliminated before HR occurs. In that context, a lot of efforts were undertaken to tightly control CRISPR-mediated gDNA cleavage, for example by implementing inducible transcriptional and translational gene expression systems (Canadas et al., 2019; Wasels et al., 2017).

Bacterial inducible systems are however prone to transcriptional leakage, which can lead to basal CRISPR gene expression levels severely impeding transformation efficiencies (Wasels et al., 2020). Our laboratory has recently developed a CRISPR-Cas9 gene editing technique for *C. acetobutylicum* based on the controlled inhibition of Cas9 gDNA cleavage by the anti-CRISPR protein AcrIIA4 from *Listeria monocytogenes* (Wasels et al., 2020), Fig. 1a). In brief, transformation is carried out in the presence of lactose, which induces AcrIIA4 expression and protects the cells from Cas9 activity. After growth, colonies are subsequently exposed to a medium devoid of lactose and containing the Cas9-sgRNA inducer anhydrotetracycline (aTc), whose expression selects cells that underwent correct HR. By tightly separating the transformation and gene editing steps, the CRISPR/anti-CRISPR method allowed efficient and iterative gene edition in *C. acetobutylicum*, with all CRISPR constructs being able 1) to yield a significant number of colonies and 2) to generate mutants at 100 % efficiency (Wasels et al., 2020).

In this work, we applied the same CRISPR/anti-CRISPR gene editing technique to assess whether it would be transposable with similar success to *C. beijerinckii*. As a model, we chose the DSM 6423 strain, because it has so far been very reluctant to forward genetic approaches (Diallo et al., 2020). Transformability has indeed only been recently demonstrated, and CRISPR-based genome editing was shown to work at extremely low efficiencies with an aTc-inducible system.

We initially chose to target the *upp* gene (CIBE_0562) coding for an

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: francois.wasels@ifpen.fr (F. Wasels).</footnote>

<footnote n="1">¹ Present address: Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria</footnote>
uracil phosphoribosyltransferase, as its disruption was shown to result in an easy-to-screen phenotype in *C. acetobutylicum* (Foulquier et al., 2019). Indeed, Upp catalyzes the conversion of uracil to UMP, and supplying the halogenated analog 5-fluorouracil (5-FU) to wild-type cells results in cell death by accumulation of toxic nucleic acids.

We, therefore, transformed *C. beijerinckii* DSM 6423 Δ*catB* Δ*pNF2* - a DSM 6423 derivative sensitive to thiamphenicol, in which the endogenous plasmid pNF2 was removed (Diallo et al., 2020) - with pCas9<sub>ind</sub> and pCas9<sub>acr</sub>, as previously described for *C. acetobutylicum* (Wasels et al., 2020, 2017). The pEC750C-Δ*upp*, a pEC750C plasmid derivative containing a sgRNA targeting *upp* and homology regions designed to delete this gene (Fig. 1b) was next generated. While *C. beijerinckii* DSM 6423 Δ*catB* Δ*pNF2* pCas9<sub>ind</sub> could not be transformed with pEC750C-Δ*upp*, the strain bearing pCas9<sub>acr</sub> was easily transformed when plated on lactose-containing medium. After aTc induction of Cas9 and cognate sgRNA, 8 colonies were picked, which showed 100 % editing efficiency (Fig. 1c), demonstrating the proof-of-concept that our

CRISPR/anti-CRISPR genome editing approach worked similarly in *C. beijerinckii* as previously described for *C. acetobutylicum*.

Next, the newly obtained Δ*upp* strain was tested for its sensitivity to 5-FU and was surprisingly shown to behave as the Wild Type (WT) strain. In fact, a closer look at metabolic pathways potentially enabling conversion of 5-FU into 5-FUMP suggested a secondary pathway was likely involved (Fig. 2a). We therefore designed new plasmids targeting genes *udp* (CIBE_3715) and *udk* (CIBE_1796), coding for a uridine phosphorylase and a uridine kinase, respectively (Fig. 2b). After curing the pEC750C-Δ*upp* from the Δ*upp* strain, we transformed the resulting *C. beijerinckii* DSM 6423 Δ*catB* Δ*pNF2* Δ*upp* pCas9*acr* with those plasmids, referred to as pNF3C-Δ*udp* and pNF3C-Δ*udk*. Like *upp* deletion, transformation worked when lactose was supplemented to the medium, and aTc induction yielded the double knock-out mutants Δ*upp* Δ*udp* and Δ*upp* Δ*udk* (Fig. 2c). Single knock-out mutants were also generated for control. A 5-FU toxicity assay further revealed that only the Δ*upp* Δ*udp* strain was completely resistant to 5-FU, while the Δ*udp* strain appeared

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Application of a CRISPR/anti-CRISPR genome editing method to *C. beijerinckii* DSM 6423. a. Underlying principles of the approach. Two plasmids are used. The first one bears invariable genetic elements: *cas9* under the control of the TetR repressor, *acrIIA4* under the control of the LacR repressor. The second one contains the sgRNA targeting cassette (also repressed by TetR) and the homology regions necessary for correct recombination. Lactose promotes the expression of *acrIIA4*, thereby inactivating *Cas9*, while anhydrotetracycline induces the expression of *Cas9* and cognate sgRNA. b. gDNA regions surrounding the *upp* gene and deletion design. c. Anhydrotetracycline induction of *acrIIA4* expression in colonies obtained after transformation yields knock-out mutants at 100 % editing efficiency.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Iterative deletion of uracil uptake pathways in *C. beijerinckii* DSM 6423. a. Resistance to 5-fluorouracil is predicted to be mediated by two distinct pathways. b. gDNA regions surrounding the *udp* and *udk* genes and deletion design. c. Single and double knock-out mutants obtained after anhydrotetracycline induction. d. 5-fluorouracil toxicity assay in single and double knock-out mutants.</figcaption></figure>

as partly resistant. That the Δupp Δudk strain did not confer resistance to 5-FU was surprising and suggests that 5-fluorouridine is likely metabolized through another pathway. Collectively, we could efficiently and iteratively delete multiple genes using our CRISPR/anti-CRISPR approach in *C. beijerinckii* DSM 6423, which, in addition to our previous results in *C. acetobutylicum* (Wasels et al., 2020), suggests that this tool could be broadly applicable to *Clostridia*.

# 1. Material & methods

## 1.1. Strains, plasmids and primers

### 1.1.1. Bacterial strains are described in Table 1

*Clostridium beijerinckii* strains were cultured in an anaerobic chamber (Bactron) at 34°C without shaking. 2YTG (per liter: 16 g tryptone, 10 g yeast extract, 5 g NaCl, 20 g glucose, pH 5.2) was used for liquid cultures. For solid media, the pH was not adjusted, agarose (15 g/L) was added, and less glucose (5 g/L) was used. When necessary, erythromycin (Sigma-Aldrich, 40 mg/L) and/or thiamphenicol (Fisher Scientific, 15 mg/L) was added.

*Escherichia coli* strains were grown in liquid or solid LB media, in

aerobic conditions at 37 °C with 180 rpm agitation when required. When necessary, erythromycin (100–500 mg/L), chloramphenicol (Fisher Scientific, 25 mg/L), tetracycline (Sigma-Aldrich, 10 mg/L) or ampicillin (Sigma-Aldrich, 100 mg/L) was added.

Plasmids and primers used in this work are described in Tables 1 and 2, respectively. The sequence of pCas9acr is available on Genbank (accession number to be determined). Vectors constructed in this study are hereafter described in dedicated sub-sections.

## 1.2. Construction of a pNF2-derived *E. coli* / *Clostridium shuttle vector*

To construct an artificial shuttle plasmid containing the pNF2 origin of replication, we first amplified most of the pNF2 plasmid (with the exclusion of CIBE_p20002–3, predicted to encode an exported and a membrane protein, respectively) with the pNF2_fwd and pNF2_rev primers. The PCR product was then inserted in the pUC19 plasmid at the *Sal*I and *Bam*HI sites by restriction cloning to obtain the pNF3 plasmid.

The *catP* gene (from *C. perfringens*; conferring thiamphenicol resistance) was next amplified from pEC750C with the *catP_fwd* and *catP_rev* primers and introduced in the pNF3 plasmid at the *Bam*HI and *Sac*I restriction sites to obtain the pNF3C shuttle plasmid.
<table><caption>**Table 1**
Bacterial strains and plasmids used in this study.</caption><thead><tr><th>Bacterial strain or plasmid</th><th>Relevant Characteristics</th><th>Source or reference</th></tr></thead><tbody><tr><td>Strains</td><td></td><td></td></tr><tr><td>C. beijerinckii DSM 6423 ΔcatB ΔpNF2</td><td>ΔcatB ΔpNF2</td><td>(Diallo et al., 2020)</td></tr><tr><td>E. coli NEB 10-beta Plasmids</td><td></td><td>NEB</td></tr><tr><td>pCas9<sub>ind</sub></td><td>ermB, ColE1 origin, pCB102 origin, cas9 (Pcm-tetO2/1 promoter), tetR</td><td>(Wasels et al., 2017)</td></tr><tr><td>pCas9<sub>acr</sub></td><td>pCas9<sub>ind</sub> derivative with acrIIA4 (Pbgal promoter) and bgaR insertions</td><td>(Wasels et al., 2020)</td></tr><tr><td>pEC750C</td><td>catP, ColE1 origin, pIP404 origin</td><td>(Wasels et al., 2017)</td></tr><tr><td>pNF3C</td><td>catP, ColE1 origin, replication origin from pNF2</td><td>This study</td></tr><tr><td>pEC750C-Δupp</td><td>pEC750C derivative targeting upp and carrying a Δupp editing template insertion</td><td>This study</td></tr><tr><td>pNF3C-Δudp</td><td>pNF3C derivative targeting udp and carrying a Δudp editing template insertion</td><td>This study</td></tr><tr><td>pNF3C-Δudk</td><td>pNF3C derivative targeting udk and carrying a Δudk editing template insertion</td><td>This study</td></tr></tbody></table>

### 1.3. Plasmids construction for upp deletion

#### 1.3.1. The pEC750C-Δupp was constructed in two steps

The upp homology regions (designed as in Fig. 1b) were first PCR amplified using primers upp_HR1_fwd / rev and upp_HR2_fwd / rev from C. beijerinckii DSM 6423 gDNA. These parts were then assembled into pEC750C linearized by Sall / SacI by Gibson Assembly (NEB), yielding pEC750C-upp-HR.

The protospacer sequencing for sgRNA-mediated targeting of upp was designed with the Geneious R10 software (www.geneious.com).

This sequence, along with a constitutive sRNA promoter (from gene sCbei_5830 of C. beijerinckii NCIMB 8052 (Chen et al., 2011)), a sgRNA scaffold and XhoI / NcoI restriction sites, was ordered as a single synthesized DNA (Eurofin Genomics) in pEX-A2. After PCR amplification with pEX_fwd and pEX_rev, the pEC750C-Δupp was obtained by inserting this PCR product by restriction cloning into pEC750C-upp-HR at the XhoI and NcoI sites.

### 1.4. Plasmids construction for udp and udk deletions

#### 1.4.1. The pNF3C-Δudp and pNF3C-Δudk plasmids were generated in several steps

Firstly, a BsaI restriction site was removed from the pNF3C vector by the “Quickchange” method using the ΔBsaI_fwd and ΔBsaI_rev primers. The sgRNA cloning cassette from pGRNA<sub>ind</sub> was next PCR amplified with primers pEC750C_fwd and M13_rev and cloned by restriction at the SacI site of the BsaI-free pNF3C to obtain the pNF3C-sgRNA<sub>ind</sub> vector.

Secondly, homology regions designed to delete target genes (Fig. 2b) were PCR amplified with primers udp_HR1_fwd / rev, udp_HR2_fwd / rev, udk_HR1_fwd / rev and udk_HR2_fwd / rev. Each pair of homology region was assembled by PCR fusion, and the resulting homology templates were introduced in pNF3C-sgRNA<sub>ind</sub> at the BamHI site by restriction cloning to obtain the pNF3C-GRNA<sub>ind</sub>-udp-HR and pNF3C-GRNA<sub>ind</sub>-udk-HR plasmids.

Lastly, sgRNA protospacer sequences targeting udp and udk coding sequences were designed with the Geneious R10 software (www.geneious.com). For each protospacer, a pair of primers (udp_guide_fwd / rev & udk_guide_fwd / rev) was annealed and introduced into the pNF3C-GRNA<sub>ind</sub>-udp-HR and pNF3C-GRNA<sub>ind</sub>-udk-HR plasmids via Golden Gate assembly (BsaI site), resulting in the pNF3C-Δudp and pNF3C-Δudk plasmids.

<table><caption>**Table 2**
Primers used in this study.</caption><thead><tr><th>Name</th><th>Sequence (5'-3')</th></tr></thead><tbody><tr><td>pNF2_fwd</td><td>TACCAGGGATCCGTATTAATGTAACTATGATATCAATTCTTG</td></tr><tr><td>pNF2_rev</td><td>ACTTGGGTCGACCACGATAAAAACAAGGTTTTAAGG</td></tr><tr><td>catP_fwd</td><td>ATGCATGGATCCCGGCAGTTTTCTTTTTCGG</td></tr><tr><td>catP_rev</td><td>ATGCGAGAGCTCGGTTAAATACTAGTTTTTAGTTACAGAC</td></tr><tr><td>upp_HR1_fwd</td><td>ACGCGTGACGTCGACATAAGGTACCAGGAATTAGAGCAGC</td></tr><tr><td>upp_HR1_rev</td><td>TCTATCTCCAGCTCTAGACCATTATTATCCTCCAAGTTTGCT</td></tr><tr><td>upp_HR2_fwd</td><td>ATAATGGTCTAGAGCTGGAGATAGATTATTTGGTACTAAG</td></tr><tr><td>upp_HR2_rev</td><td>TATGACCATGATTACGAATTCGAGCTCGAAGCGCTTATTATTGCATTAGC</td></tr><tr><td>pEX_fwd</td><td>CAGATTGTACTGAGAGTGCACC</td></tr><tr><td>pEX_rev</td><td>GTGAGCGGATAACAATTTCACAC</td></tr><tr><td>ΔBsaI_fwd</td><td>GTGGGTCACGCGGTATCATTGC</td></tr><tr><td>ΔBsaI_rev</td><td>GCAATGATACCGCGTGACCCAC</td></tr><tr><td>pEC750C_fwd</td><td>CAATATTCCACAATATTATATTATAAGCTAGC</td></tr><tr><td>M13_rev</td><td>CAGGAAACAGCTATGAC</td></tr><tr><td>udp_HR1_fwd</td><td>ATCATAGTTACATTAATACGGATCCGGAGTATGTTTTTCTAATAATTCAAC</td></tr><tr><td>udp_HR1_rev</td><td>GTAGGTTCAGTATTTCTAGTTGTTG</td></tr><tr><td>udp_HR2_fwd</td><td>CAACAACTAGAAATACTGAACCTACTATAATACCTCCATTTATTTCACG</td></tr><tr><td>udp_HR2_rev</td><td>CGAAAAAGAAAAACTGCCGGGATCCGGTAATCTACTTCCTTCTGG</td></tr><tr><td>udk_HR1_fwd</td><td>CGAAAAAGAAAAACTGCCGGGATCCCTTTCCAATAATTCAAGAGC</td></tr><tr><td>udk_HR1_rev</td><td>AATTATATTAGATTACTTATGGAGGTAGCAGTACATAATTTTTAAGTAG</td></tr><tr><td>udk_HR2_fwd</td><td>CCTCCATAAGTAATCTAATATAATTATAC</td></tr><tr><td>udk_HR2_rev</td><td>ATCATAGTTACATTAATACGGATCCGCTTTAAAAAATATACTTTCAGG</td></tr><tr><td>udp_guide_fwd</td><td>TCATGTAAATACGTAATATTACCA</td></tr><tr><td>udp_guide_rev</td><td>AAACTGGTAATATTACGTATTTAC</td></tr><tr><td>udk_guide_fwd</td><td>TCATCGTTGATCCGAGTAAGAGAA</td></tr><tr><td>udk_guide_rev</td><td>AAACTTCTCTTACTCGGATCAACG</td></tr><tr><td>upp_fwd</td><td>CGGATATTGCATTACCAGTAGC</td></tr><tr><td>upp_rev</td><td>TTATCAATCTCTTACACATGGAGC</td></tr><tr><td>udp_fwd</td><td>AAAGGACATTGGTAATAATTCTTCG</td></tr><tr><td>udp_rev</td><td>CACCAATTAATTTAGATAGTTCAGG</td></tr><tr><td>udk_fwd</td><td>GTAAAGTGGGTATATGTTAAATG</td></tr><tr><td>udk_rev</td><td>TCAACTTGATCTCCACAAAC</td></tr></tbody></table>
## 1.5. Bacterial transformation and genome editing

Clostridium beijerinckii DSM 6423 ΔcatB was transformed as previously described (Diallo et al., 2020) with plasmids produced from a *dam* dcm* strain (INV110, Invitrogen). For electroporations of sgRNA / homology regions-containing plasmids into Clostridium beijerinckii DSM 6423 ΔcatB (pCas9acr), lactose (40 mM) was added at each step in all culture media.

For strains containing both pCas9acr and a sgRNA / homology region plasmid, genome editing was induced by spotting single colonies onto plates containing 200 ng/mL anhydrotetracycline (Sigma-Aldrich) and no lactose, similarly to what was described before (Wasels et al., 2017). Individual anhydrotetracycline-resistant colonies were further tested for successful gene deletion.

## 1.6. Verification of gene deletions and phenotypic assays

Colony PCR was performed to test whether gene editing successfully occurred. Primers (upp_fwd / rev, udp_fwd / rev or udk_fwd / rev) were designed to amplify a genomic region encompassing the homology regions used to delete the targeted genes, as described in Figs. 1c and 2c. For C. beijerinckii DSM 6423, colony treatment was adapted for reproducibility and consistency. Fresh colonies were suspended in 100 µL TE and treated 10 min at 99°C without agitation. 900 µL deionized water was then added, and the lysates were centrifuged at 20,000 g for 3 min. 0.5 µL of the supernatant was then used for 10 µL PCR reactions using Phusion DNA polymerase (ThermoFischer). PCR products are then visualized by agarose gel electrophoresis.

The sensitivity of mutants to 5-FU (Sigma-Aldrich) was tested by streaking them onto 2YTG plates containing 50 mg/L 5-FU and growing them anaerobically for 72 h at 34°C.

### CRediT authorship contribution statement

**Nicolas Lopes Ferreira:** Supervision, Resources, Project administration, Funding acquisition, Conceptualization. **François Wasels:** Writing – review & editing, Supervision, Project administration, Conceptualization. **Hocq Remi:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. **Gwladys Chartier:** Investigation.

### Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Francois Wasels has patent #FR3090691B1 issued to IFP Energies Nouvelles. Francois Wasels has patent #FR3096373B1 issued to IFP Energies Nouvelles. Francois Wasels has patent #FR3111642B1 issued to IFP Energies Nouvelles. Francois Wasels has patent #FR3081881B1 issued to IFP Energies Nouvelles. Remi Hocq has patent #FR3090691B1 issued to IFP Energies Nouvelles. Remi Hocq has patent #FR3096373B1 issued to IFP Energies Nouvelles. Remi Hocq has patent #FR3111642B1 issued to IFP Energies Nouvelles. Nicolas Lopes Ferreira has patent #FR3090691B1 issued to IFP Energies Nouvelles. Nicolas Lopes Ferreira has patent #FR3096373B1 issued to IFP Energies Nouvelles. Nicolas Lopes Ferreira has patent #FR3111642B1 issued to IFP Energies Nouvelles. Nicolas Lopes Ferreira has patent #FR3081881B1 issued to IFP Energies Nouvelles. Gwladys Chartier has patent #FR3090691B1 issued to IFP Energies Nouvelles. Gwladys Chartier has patent #FR3081881B1 issued to IFP Energies Nouvelles. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

### Data availability

Data will be made available on request.

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