A universal chassis plasmid pwh34 was first constructed to facilitate the

Transcription

1 File name: Supplementary method Plasmids construction A universal chassis plasmid pwh34 was first constructed to facilitate the construction of plasmids for targeting and editing of specific genes. The constructed pwh34 contains following components: i) NdeI linearized pmtl82151 backbone plasmid; ii) the Cpf1 coding sequence drove by the lactose inducible promoter (ilacp::cpf1) (1); iii) the terminator of the thiolase gene (thlt) from C. beijerinckii NCIMB 8052, and iv) an 40-bp synthetic DNA sequence for easy programing of the crrna for gene targeting. Briefly, ilacp was amplified from pyw34-btgzi using primers YW2817/YW2818 (2). The E. coli codon optimized Cpf1 gene (from Acidaminococcus sp. BV3L6; AsCpf1) was amplified from pdest-hismbp-ascpf1- EC (Addgene) using primers YW2819/YW2820 (3). The resultant ilacp and Cpf1 gene fragments were assembled into NdeI restriction site of pmtl82151 to generate pwh34s. Then thlt was amplified from C. beijerinckii genome using primers YW2823/YW2824 (4). The resultant PCR amplicon contained two oppositely oriented BtgZI sites, making it convenient to incorporate crrna sequences to target on specific chromosomal loci. The PCR amplicon was inserted into the BamHI site of pwh34s, generating pwh34. The designated CRISPR-Cpf1 vector targeting on a specific locus would have the spacer sequence enfolded between two repeat sequences (that is, repeat-spacerrepeat ). In order to readily change the spacer sequence to target onto a new gene locus, we constructed the plasmid pwh37 serving as the PCR template to generate the new pre-crrna ( repeat-spacer-repeat ). It contains only repeat-spacer (rather than repeat-spacer-repeat ) to avoid the potential false priming in the PCR reaction. To construct pwh37, the small RNA promoter (srnap; promoter of scbei_5830) 1

2 26 27 was amplified from C. beijerinckii 8052 genome with primers YW2821/YW2822 (2), and meanwhile the crrna sequence (5-28 TAATTTCTACTCTTGTAGATCATCTGATAAGAAGGACTTAATA-3 ) was added to the 3 -end of the PCR amplicon. The hybrid PCR amplicon was assembled to the BamHI site of pwh34, generating pwh37. pwh41s is a derivative from pwh37 containing the crrna to target on the ferric uptake regulator (fur) gene (but without the homology arms flanking fur for homologous recombination). To construct pwh41s, srnap::crrna-fur was amplified with primers YW3105/YW3106, in which the interchangeable spacer within pwh37 was replaced with the specific spacer 5 - GCTCAGCTAACGGAACATGGCTT-3 (PAM sequence: 5 -TTTA-3 ) targeting on fur. The resultant PCR amplicon was assembled with the BtgZI-linearized pwh34 to generate pwh41s. Further, the other plasmids designated for gene editing (including pwh41, pwh55, pwh74. pwh75, pwh79 pwh173 and pwh174) were all constructed by the One-Step-Assembly (OSA) approach. The gene context information for each target gene is illustrated in Fig. 4a. All the primers for constructing single-genetargeting plasmids were designed by OPF algorithm (see the next section). The GC content of spacers was set between 39%-52%, and the length of homology arms and the overlap region for assembly were set to ~500 bp and 25 bp, respectively. The plasmid pwh41 was designed to delete the 390-bp fur gene (CD630_08260) in C. difficile 630. Briefly, the srnap::crrna-fur fragment was amplified using primers YW3105/YW3713, in which the interchangeable spacer in pwh37 was replaced with the specific spacer 5 -GCTCAGCTAACGGAACATGGCTT-3 (PAM sequence: 5 - TTTA-3 ) targeting on fur. The 500-bp homology arms fur-up-arm and fur-down- 2

3 arm flanking fur were amplified from the C. difficile 630 genomic DNA (gdna) with primers YW3714/YW3715 and YW3716/YW3717, respectively. These three fragments cited above (srnap::crrna-fur, fur-up-arm and fur-down-arm) were assembled with the BtgZI-linearized pwh34 in an OSA manner to generate pwh41. Primers YW3718/YW3719 were designed for the detection of fur deletion. The PCR amplicons of the wild-type (WT) strain and the fur deletion mutant were 2,290 bp and 1,900 bp, respectively. The plasmid pwh55 was designed to delete the 1,833-bp cwp66 gene (CD_27890, encoding a cell wall protein). Similarly as constructing pwh41, the srnap::crrna-cwp66 fragment, with the specific spacer 5 - GCAGTGGGTGTATTAGCAGCTAA-3 (PAM sequence: 5 -TTTA-3 ), was amplified with primers YW3105/YW3304. Homology arms cwp66-up-arm and cwp66-down-arm were amplified from the C. difficile 630 gdna with primer pairs of YW3305/YW3306 and YW3307/YW3308, respectively. The three fragments generated above were assembled with BtgZI-linearized pwh34 to generate pwh55. Primer pair YW2369/YW2370 was designed to detect the deletion of cwp66. The PCR amplicons of WT and cwp66 deletion mutant were 3,180 bp and 1,350 bp, respectively. The plasmid pwh74 was designed to delete the 1,920-bp tetracycline resistance protein (tetm) gene (CD630_05080). The srnap::crrna-tetm fragment, with specific spacer 5 -CGGGATTCGGTTAGAATATCGGA-3 (PAM sequence: 5 -TTTG-3 ), was generated with primers YW3105/YW3456. Homology arms tetm- Up-arm and tetm-down-arm were amplified from the C. difficile 630 gdna with primer pairs of YW3457/YW3458 and YW3459/YW3460, respectively. The three fragments generated above were assembled with BtgZI-linearized pwh34 to generate 3

4 pwh74. Primer pair YW3461/YW3462 was used to detect the deletion of tetm. The PCR amplicons of the WT strain and the tetm deletion mutant were 3,106 bp and 1,200 bp, respectively. The plasmid pwh75 was designed to delete a 3,145-bp gene locus, containing ermb1 (CD630_20070), putative hydrolase (CD630_20080) and ermb2 (CD630_20100). In a similar manner, srnap::crrna-ermb1/2, with specific spacer 5 -GAAAGCCGTGCGTCTGACATCTA-3 (PAM sequence: 5 -TTTT-3 ), was generated with primers YW3105/YW3463. Fragments ermb1/2-up-arm and ermb1/2-down-arm were generated with primer pairs of YW3464/YW3465 and YW3466/YW3467, respectively. Then the three fragments were assembled with BtgZI-linearized pwh34 to generate pwh75. The primer pair YW3468/YW3469 was used to detect the deletion of the target loci. The PCR amplicons of the WT strain and the ermb1/2 deletion mutant were 4,505 bp and 1,360 bp, respectively. Multi-gene editing plasmid pwh79 was designed to delete cwp66 and tcda (CD630_06630, encoding toxin A) simultaneously. The fragment srnap::crrnacwp66-tcda, with cwp66 and tcda specific spacers (5 - GCAGTGGGTGTATTAGCAGCTAA-3 and 5 - AGTACCTCAGTTAAGGTTCAACT-3, respectively), was generated with primers YW3105/YW3529. Homology arms cwp66-up-down-arms were amplified from pwh55 with primers YW3530/YW3531. While homology arms tdca-up-arm and tcda-down-arm were amplified from the C. difficile 630 gdna with primers YW3532/YW3533 and YW3534/YW3535, respectively. The four fragments including srnap::crrna-cwp66-tcda, cwp66-up-down-arms, tdca-up-arm and tcda-down-arm were assembled with BtgZI linearized pwh34 to generate pwh79. Primer pair YW2369/YW2370 was used to detect the deletion of cwp66 as mentioned 4

5 above. Primer pair YW3321/YW3322 was designed to detect the deletion of tcda. The PCR amplicons of the WT strain and the tcda deletion mutant were 9,625 bp and 1,500 bp, respectively. The plasmid pjz173 was designed to delete the ~49.2-kb phicd630-2 locus CD630_28890-CD630_29520). The fragment srnap::crrna-phicd630-2, containing the specific spacer 5 -TACATCAAGCTCAAAGTCACTCC-3 (PAM sequence: 5 -TTTT-3 ), was generated with primers YW3105/YW3333. Homology arms phicd630-2-up-arm and phicd630-2-down-arm were generated with primers YW3334/YW3335 and YW3336/YW3337, respectively. Then the three fragments were assembled with BtgZI-linearized pwh34 to generate pjz173. The plasmid pjz174 was also designed to delete the ~49.2-kb phicd630-2 locus, however, with two spacers (5 -TACATCAAGCTCAAAGTCACTCC-3 and 5 - GTAACCTATGGGAATTGTACCAC-3 ) in order to improve DSB efficiency for this extremely large gene deletion. The fragment srnap::crrna-phicd630-2-double was generated with primers YW3105/YW3338, in which a second spacer 5 - GTAACCTATGGGAATTGTACCAC-3 was added to the 3 -end of srnap::crrna-phicd The rest of the plasmid construction process was similar to the construction of pjz173. The plasmid pjz180 was constructed for the attempt to delete phicd630-2 in C. difficile using the Type II CRISPR-Cas9 system derived from S. pyogenes. The plasmid pyw34-btgzi was used as the mother vector (2), which contains a Cas9 open reading frame (ORF) driven by the lactose inducible promoter and a chimeric grna sequence (2). First, the plasmid pjz23-cas9 was created from pyw34-btgzi through Gibson Assembly as follows. The CAK1 replicon and erythromycin (Erm) marker of pyw34-btgzi were replaced with pbp1 replicon and thiamphenicol (Tm) 5

6 marker, respectively, through an in vitro digestion with Cas9 nuclease following the protocol as described previously (2), generating plasmid pyw34-pbp1-tm-btgzi. The pbp1 replicon and the Tm marker were amplified from pmtl82151 using primers YW1073/YW1074 and YW1029/YW1030, respectively. The TraJ fragment which is essential for the conjugation process was also amplified from pmtl82151 using primers YW1060/YW1061 and then inserted into the ApaI site of pyw34- pbp1-tm-btgzi through Gibson Assembly, generating pjz23-cas9. Based on pjz23- Cas9, the srnap (2) fused with the 20-nt guiding sequence (5 - TACGATTCTAGCACTGCTGC-3 ) targeting on phicd630-2 and two homology arms (~1 kb each) were cloned into the BtgZI and NotI sites, respectively, as described previously (5), generating pjz180. Primer pair YW3342/YW3343 was designed to detect the deletion of phicd The PCR amplicon of phicd630-2 deletion mutant was 2,471 bp, whereas the phicd630-2 region in the WT strain is too long to be amplified using primers YW3342/YW3343. Therefore, additional primers YW3342/YW3799 were designed to amplify the junction between phicd630-2-up-arm and phicd630-2 gene for a further confirmation. It should be noticed that, to enhance the recombination efficiency for deleting this extremely large fragment, homology arms of ~1000 bp (instead of ~500 bp) were used for all three phicd630-2-targeting plasmids (pjz173, pjz174 and pjz180) (6)

7 References: 1. Hartman AH, Liu H, Melville SB Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression in Clostridium perfringens. Appl Environ Microbiol 77: Wang Y, Zhang ZT, Seo SO, Lynn P, Lu T, Jin YS, Blaschek HP Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable clean Mutant Selection in Clostridium beijerinckii as an Example. ACS Synth Biol 5: Hur JK, Kim K, Been KW, Baek G, Ye S, Hur JW, Ryu SM, Lee YS, Kim JS Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol 34: Gao W, Long L, Tian X, Xu F, Liu J, Singh PK, Botella JR, Song C Genome Editing in Cotton with the CRISPR/Cas9 System. Front Plant Sci 8: Wang S, Dong S, Wang P, Tao Y Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 system 83: Faulds-Pain A, Wren BW Improved bacterial mutagenesis by highfrequency allele exchange, demonstrated in clostridium difficile and streptococcus suis. Appl Environ Microbiol 79: