sgRNA design and vector construction
Guide sequences for two sgRNAs (sgRNA16–1, sgRNA16–2) targeting intron 16 and two sgRNAs (sgRNA17–1, sgRNA17–2) targeting intron 17 of the porcine KIT gene were selected using an open tool: CRISPR DESIGN (https://benchling.com/crispr). The oligos of each sgRNA guide sequence were cloned downstream of the human U6 promoter via Bbs I restriction sites in the plasmid pSpCas9(BB)-2A-GFP (pX458) (Addgene plasmid #48138) to create the plasmid pX458-sgRNA. Positive clones were confirmed by Sanger sequencing (Sangon, China). sgRNA sequences and details were listed in Additional file 1: Table S1.
Porcine kidney cell culture, transfection, and sorting
Two New born Yorkshire piglets were purchased from Guangxi yangxiang Technology Co., Ltd. (China). After sacrificing these piglets, porcine kidney cells were isolated from kidneys and cultured in Dulbecco’s modified Eagle medium (Gibco, USA) supplemented with 100 units ml− 1 penicillin, 100 μg ml− 1 streptomycin (Gibco, USA), and 10% foetal bovine serum (Gibco, USA) at 37 °C under a 5% CO2 humidified atmosphere (Thermo, USA). The animal study was supervised the Institutional Animal Care and Use Committee of the Sun Yat-sen University (approval no. IACUC DD-17-0403) and used in accordance with regulation and guidelines of this committee. For electroporation, porcine kidney cells were harvested and counted, and 1 × 106 cells were resuspended in 100 μl buffer R (Invitrogen, USA), containing 10 μg pX458-sgRNA plasmid. The mixture was then transfected through electroporation at 1650 V for 10 ms in 3 pulses using the Neon transfection system (Invitrogen, USA) and seeded into 6-well plates (Nunc, USA) with 2 ml preheated culture medium. After 24 h of transfection, the culture medium was refreshed gently to exclude dead cells. Cells were then observed and photographed with a fluorescence microscope (Nikon, Japan). After 48 h of transfection, cells were dissociated with trypsin (Sigma, USA) at 37 °C for 4 min and resuspended in PBS (Gibco, USA), then analysed and collected by fluorescence-activated cell sorting (FACS) using Aria II cell sorter (BD Biosciences, USA). EGFP-positive cells were sorted into 1.5-ml centrifuge tubes and centrifuged either for further culturing or used for the isolation of genomic DNA. The single cell was seeded into 96-well plates using Aria II cell sorter. After three weeks of culture, the single cell was expanded for subsequent analysis.
T7E1 assay and Nla III assay
Genomic DNA samples were extracted from EGFP positive cell populations using the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s instructions. The targeted sites were amplified by PrimerSTAR HS DNA polymerase (TaKaRa, Japan) with the primer pairs and purified with a gel extraction kit (Omega, USA). Then, 300 ng purified PCR products for T7 endonuclease I (T7E1) assay were denatured and annealed in NEBuffer 2 using a thermocycler (Bio-Rad, USA), then digested with T7E1 (NEB, UK) for 30 min at 37 °C and separated by 10% native polyacrylamide gel electrophoresis (native-PAGE). Mutation frequencies were calculated based on the band intensities using Image J software and then PCR products were cloned into a pMD-18 vector (Takara, Japan) and sequenced to confirm the mutation efficiency by dividing the number of mutant clones by the number of total clones. Primers used for PCR are listed in Additional file 1: Table S2.
The G > A mutation in the first base of intron 17 of KIT introduces the restriction site Nla III. We amplified a 145 bp fragment across the splice mutation site and digested the PCR products using the Nla III enzyme to determine the efficiency of the deletion of KIT copies with G > A mutation by CRISPR/Cas9 (Fig. 3a). A complete deletion of KIT copies with the G > A mutation would eliminate the restriction site, which is detected as a failure to cleave the PCR product by Nla III. In contrast, a complete deletion of a normal KIT copy would result in complete digestion of the PCR product by Nla III. Purified PCR products for Nla III assay were amplified and digested with Nla III (Thermo, USA) for 5 min at 37 °C and separated by 15% native-PAGE. The primers used for PCR are listed in Additional file 1: Table S3.
Real-time quantitative PCR (qPCR) analysis
Copy number variation was estimated using real-time quantitative PCR and the 2-△△CT method as described by Livak and Soejima [13, 22]. The primers were designed using Primer-BLAST on NCBI and the primer details for KIT (Genbank accession number: CU929000.2) and COL10A1 (Genbank accession number: AF222861.1) are listed in (Additional file 1: Table S4). The copy number of c-kit was normalized against the Col10 region, a control region in the genome that did not vary in copy number between the pigs . The PCR reaction was performed using the Roch LC480 in 20 μl reaction volumes using ChamQ™ SYBR qPCR Master Mix (Vazyme, China). The procedure in the thermal cycling was an initial 5 min hold at 95 °C, followed by 40 cycles of 15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C.
To determine the site-specific cleavage of the CRISPR/Cas9 system in vitro, potential off-target sites (Additional file 1: Table S5) were evaluated by CRISPR DESIGN (https://benchling.com/crispr). Each five top-scoring off-target sites of sgRNA16–1 or sgRNA17–6 were selected for the T7E1 assay (Additional file 1: Table S6) and those yielding typical cleavage bands were considered as candidates. Finally, the PCR products of the candidates were sequenced to confirm the off-target effects. Further confirmation of the targeting specificity of sgRNA17–6 was carried out by analysing each five off-target sites with high, medium, or low scores (Additional file 1: Table S5) by T7E1 assay (Additional file 1: Table S6).