File name: Supplementary Information. Description: Supplementary Figures, Supplementary Tables and Supplementary References.

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1 1 2 File name: Supplementary Information Description: Supplementary Figures, Supplementary Tables and Supplementary References. 3 1

2 4 Supplementary Figures Figure S1 Comparison of the One-Step-Assembly (OSA) method with the conventional two-step method for plasmid construction for genome editing using CRISPR-Cpf1 or 2

3 CRISPR-Cas9. (a) A flow-chart of the One-Step-Assembly (OSA) method. Briefly, the gene-specific srnap::crrna, ~500-bp homologous Up-arm and ~500-bp homologous Down-arm are generated through PCR. These three fragments are assembled with BtgZI-linearized pwh34 in a One-Step-Assembly (OSA) manner to construct the gene-targeting plasmid within two days. In addition, a Python-based algorithm has been developed to help design primers in an automatic fashion to facilitate the OSA procedure. (b) A flow-chart of the conventional two-step method. Briefly, the gene-specific srnap::crrna is firstly inserted into BtgZI sites of the chassis plasmid; then ~500-bp homologous Up-arm and ~500-bp homologous Down-arm are assembled to the NotI site of the plasmid that is obtained in the first step. Because two cycles of Gibson assembly, screening, culturing, and plasmid extraction processes are required in this two-step method, it takes at least four days to generate the desirable gene-targeting plasmid. 3

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5 Figure S2 Design primers using the OSA Primer Finder (OPF) for the construction of the gene-targeting CRISPR-Cpf1 plasmid (taking the plasmid for fur deletion as an example). The fur gene sequence along with the upstream ~1000 bp and downstream ~1000 bp is first downloaded from NCBI (Accession number: AM ). Before launching OPF, Python 2.7 ( should be pre-installed on the computer. Then, the OPF program can be launched by double-clicking the OPF.py file (Supplementary file1). The fur gene sequence can then be input into the appropriate blank as indicated by the algorithm. Then the User will be asked whether he/she prefers to identify protospacer sequences on the Sense or antisense strand. After inputting the answer and pressing the enter key, all potential protospacers in either sense or antisense strand (whichever has been selected by the User) of fur gene will be listed. At this point, the Primer 0 corresponding to each protospacer will be generated and listed underneath the protospacer. Furthermore, the position (site; sense/antisense), reverse complementary sequence (RVS), GC content (GC%) and T m value (T m ) for each protospacer will also be determined. Then, the User can select one specific protospacer to use based on his/her own criteria (GC%, site, etc.). After inputting the Up-arm sequence and Down-arm sequence and defining the Length of overlap regions of adjacent fragments (the region on the homology arm that is covered by the primer; the primers are designed to amplify fragments with extended sequence for the Gibson assembly purpose), the associated Primers 1, 2, 3, 4 will be generated. The primer pairs of 0/YW3105 (the universal primer), 1/2 and 3/4 will be used to generate ilacp::crrna, upstream arm and downstream arm, respectively. These three fragments can then be assembled with the BtgZI-linearized pwh34 to generate the corresponding plasmid targeting on the specific gene (fur here) through OSA. 5

6 Figure S3 Antibiotics susceptibility of C. difficile strains. The WT C. difficile 630, ermb1/2, tetm, and ermb1/2 tetm strains were streaked on BHIS, BHIS-Erm and BHIS-Tet plates. Pictures were taken after 24 h of incubation anaerobically at 37 o C. BHIS-Tet: BHIS medium supplemented with 15 µg/ml tetracycline; BHIS-Erm: BHIS medium supplemented with 50 µg/ml erythromycin. 6

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8 Figure S4 Sanger sequencing results confirming the mutation in (a) fur, (b) tetm, (c) phi CD630-2, (d) cwp66, and (e) tcda mutants. The primers used for sequencing were the same as those used for the detection using cpcr (Table S2). Sequencing data were visualized using the Chromas software (Technelysium). The Up-arm & Down-arm sequences near the conjunction site were listed. The orange box represents the ORF of each target gene on the C. difficile chromosome. The black arrow indicates the Up-arm and Down-arm junction site. The sequencing results confirmed that all the target genes (except for the erm1/2 mutant) have been precisely deleted as designated. We were not able to obtain high quality sequencing results for the erm1/2 mutant, and thus we did not include its sequencing data in this figure. However, we double checked the antibiotic susceptibility of the erm1/2 mutant, and the result 8

9 69 confirmed that the mutant was highly sensitive to erythromycin. 9