Table 1 Strains and plasmids used in this work

Strains

E. coli 10-β

High efficiency strain ideal for cloning

C3019, NEB

E. coli S17-1

Conjugative donor strain

ATCC 47,055

C. sporogenes WT

Wild type strain, Clostridium sporogenes NCIMB 10,696

NCIMB culture collection (UK)

C. butyricum WT

Wild type strain, Clostridium butyricum DSM10702

DSMZ-German Collection

C. butyricumΔpyrE

C. butyricum with deletion of the PyrE loci

[28]

C. butyricumINpyrE::ESO

C. butyricum with integration of the miniPc.b_fU-NY-ESO-1 into the PyrE loci

This work

C. butyricumINpyrE::CDP-ESO

C. butyricum with integration of the miniPc.b_fU-CDP-NY-ESO-1 into the PyrE loci

This work

C. sporogenes-NT

Non-toxic C. sporogenes NCIMB 10,696, via deletion of the putative Streptolysin S operon

[18]

C. sporogenes-NTΔpyrE

C. sporogenes-NT with deletion of the PyrE loci

This work

C. sporogenes-NTINpyrE::ESO

C. sporogenes-NT with integration of the miniPc.sp_fU-NY-ESO-1 into the PyrE loci

This work

C. sporogenes-NTINpyrE::CDP-ESO

C. sporogenes-NT with integration of the miniPc.sp_fU-CDP-NY-ESO-1 into the PyrE loci

This work

C. sporogenes-NTINpyrE::S1

C. sporogenes-NT with integration of the miniPc.b_fU-S1 into the PyrE loci

This work

C. sporogenes-NTINpyrE::CDP-S1

C. sporogenes-NT with integration of the miniPc.b_fU-CDP-S1 into the PyrE loci

This work

Plasmids

pGG2121

Golden Gate assembling and E. coli - Clostridium shuttle vector, Cm^r

[27]

pPromoters-SpnprM3-NY-ESO-1

pGG2121 ligated with promoters, the nprM3 signal peptide, and the codon-optimized NY-ESO-1 gene

This work

pPromoters-SpnprM3-CDP-NY-ESO-1

pGG2121 ligated with promoters, the nprM3 signal peptide, and the codon-optimized CDP sequence and NY-ESO-1 gene

This work

pPromoters-SpnprM3-S1

pGG2121 ligated with promoters, the nprM3 signal peptide, and the codon-optimized Spike_S1 sequence

This work

pPromoters-SpnprM3-CDP-S1

pGG2121 ligated with promoters, the nprM3 signal peptide, and the codon-optimized CDP and Spike_S1 gene sequence

This work

pPIPL12-FnCas12a

Entry vector with tetracycline-Inducible expression of FnCas12a in E. coli and clostridia, Erm^r

[28]

pPfetFn-Target_v2

Entry vector with gRNA expression for FnCas12a in E. coli and clostridia, Cm^r

[28]

pPfetFn-IncbPyrE::ESO

pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.b_fU-NY-ESO-1 expression cassette into C. butyricum PyrE loci

This work

pPfetFn-IncbPyrE::CDP-ESO

pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.b_fU-CDP-NY-ESO-1 expression cassette into C. butyricum PyrE loci

This work

pPfetFn-DspPyrE

pPfetFn-Target_v2 ligated with the donor DNA template and target for deleting pyrE gene in C. sporogenes-NT

This work

pPfetFn-InspPyrE::ESO

pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.sp_fU-NY-ESO-1 expression cassette into C. sporogenes-NT PyrE loci

This work

pPfetFn-InspPyrE::CDP-ESO

pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.sp_fU-CDP-NY-ESO-1 expression cassette into C. sporogenes-NT PyrE loci

This work

pPfetFn-InspPyrE::S1

pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.b_fU-S1 expression cassette into C. sporogenes-NT PyrE loci

This work

pPfetFn-InspPyrE::CDP-S1

pPfetFn-Target_v2 ligaed with the donor DNA template and target for integrating the miniPc.b_fU-CDP-S1 expression cassette into C. sporogenes-NT PyrE loci

This work