Pmd19 vector

We used a pKH6 vector from Dr. Stephen Lory of Harvard Medical School^{22 (link)} to construct pKH6-CRISPR2 and pKH6-CRISPR1 plasmid. These plasmids were each transfected into PA14 containing pKH-t4rnl1, respectively. Overnight cultures of PA14 WT with pKH13-t4rnl1 (or pKH13-t4K99N) and pKH6-CRISPR2 (or pKH6-CRISPR1) plasmid were grown in LB broth with antibiotic and diluted the overnight culture to OD₆₀₀ = 0.01. When OD₆₀₀ = 0.5, IPTG was added for 1 h incubation and then added the l-arabinose to 20% for different time points. The cells were centrifuged at 12,000 rpm/min for RNA isolation with DNase treatment. One microgram of total RNA was converted to complementary DNA (cDNA) was synthesized with SuperScript III First-Strand Synthesis system (Invitrogen). RT-PCR were performed using GoTaq Green Master Mix (Promega) with specific primers (Supplementary data 3). The PCR products were recovered and cloned into a pMD-19 vector (Takara) for sequencing.

Genomic DNA was extracted from transgenic and untransformed control hair roots using the modified CTAB extraction method as described previously [36] . Putative transgenic hair roots were screened preliminarily to confirm the presence of the transgenes by PCR method [37] (link). Two gene-specific primers pCa-F and pCa-R (Table 1), which flanked two loxP sites, were designed for detection of transgene excision. PCR was conducted at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, and a final extension step at 72°C for 10 min. The PCR products were loaded on 1% (w/v) agarose gel and visualized after ethidium bromide staining. The PCR fragment was cloned into pMD19 vector (TaKaRa, Dalian, China) and sequenced by Beijing Genomics Institute.

Using T-A cloning technique, the purified product (M1 and M2 segments) was cloned into pMD-19 vector (TaKaRa Biotechnology Co.,Ltd. Dalian, China), transformed into the E.coli competent cells DH5α, and the positive recombinant plasmids were screened by AMP/IPTG/X-gal, and restriction enzyme digestion and PCR identification.

Ck1α sequences from planarian Dugesia japonica species were identified in the planarian transcriptome [50 (link)]. A pair of specific primers (Djck1αF and Djck1αR) were designed to amplify the Djck1α from cDNA (extracted from intact planarians). The Djck1α sequence was cloned into PMD-19-Vector (Takara, Kyoto, Japan) for further experiments. All primers used for Djck1α and Djslit cloning and dsRNA generation are listed in Table S1.

The sgRNAs targeting Fiber-2 of FAdV-4 were designed using the guide RNA designing website,¹ introduced with BsmBI restriction sites, and cloned into the sgRNA expression vector lentiCRISPR v2. To construct the donor plasmid, the two consecutive Loxp sequences were firstly synthesized and cloned into the pUC57 vector. The RFP gene with poly(A) signal under the promoter of EF-1α was then amplified, and cloned into the vector between the two loxp sequence. Then, the Fiber-2 of FAdV-4, flanked with 1 kb left homologous arm and 1 kb right homologous arm at both sides, after introduced with sgRNA recognition sequence at both ends, was amplified and cloned into pMD19 vector (Takara, Dalian, China). Subsequently, the RFP expression cassette flanked with loxp sequence was amplified, and cloned into the site between the fiber-2 and right homologous arm through one-step cloning. The primers used for guide RNA cloning and constructing donor plasmid are listed in Tables 1, 2, respectively.

The DNA fragments containing the floR and the cfr genes were amplified by PCR using the primers (Table 1). The complete ORF fragment of the PCR product was cloned into a pMD19 vector (TaKaRa, Dalian, China). The recombinant clones were picked and sequenced. The recombinant plasmid was digested with restriction endonucleases, and the ORF fragment was recovered and further cloned into a pET28a vector (TaKaRa, Dalian, China). Finally, the recombinant plasmids were transformed into the host strain BL21. The minimal inhibitory concentrations of antibiotics were determined by the agar dilution method for the recipient strains BL21, BL21[pET28a-floR], and BL21[pET28a-cfr] in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2017).

Hctra-2 was derived from the transcriptome library. First, we verified the correctness of partial Hctra-2 sequence. Total RNA was extracted from frozen tissues with RNA prep pure tissue Kit (Tiangen, China). The cDNA was synthesized according to the manufacturer’s protocol of the PrimeScript RT reagent Kit with gDNA Eraser kit (TaKaRa, Japan). Using Tra-F and Tra-R (Table 1) as primers, the sequence of Hctra-2 was amplified by PCR under the following conditions: 94°C for 3 min, followed by 35 cycles of 95°C for 30 s denaturation, 55°C for 30 s of annealing, and 72°C for 2 min extension. After PCR, the amplified targeted-DNA was subcloned into the pMD19 vector (TaKaRa, Japan) for sequence confirmation. The full-length cDNA sequence of Hctra-2 was amplified by the SMARTer RACE 5'/3' Kit (Clontech, United States). The primers used for race amplification were T2-3' and T2-5', and the sequences are shown in Table 1. The reaction products obtained from the RACE kit instructions were purified by 1% agarose gel electrophoresis and connected to the carrier pMD19 vector and then transformed into Escherichia coli DH5α competent cells. Finally, the positive clones were screened for sequencing. Splicing 3' and 5' sequences to obtain the full-length sequence of Hctra-2.