Goat tissue samples and primary myoblast cells
The goats were raised in strict accordance with the guides of Northwest Agricultural and Forestry University (NWAFU) Animal Care and Use Committee. Heart, liver, spleen, lung, kidney, and muscle tissue samples were collected from three lamb goat (90 days old) and three adult goats (2 years old). Primary myoblast cells were isolated from skeletal muscle tissues of the lamb goat, and were purified and cultured according to previously described protocol [26]. Briefly, cells were cultured in growth medium containing DMEM/F12 basic medium (C11330500BT, Invitrogen Corp, Waltham, MA, USA), 20% fetal bovine serum (FBS) (10099-141C, Invitrogen Corp., Waltham, MA, USA), and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) (15140-122, Invitrogen Corp., Waltham, MA, USA) in 5% CO2 at 37℃, with the medium changed every day.
RNA Isolation and real-time quantitative PCR (RT-qPCR)
Total RNA was extracted from goat tissue samples or cultured cells using the reagent Trizol (9109, Takara). The concentration of total RNA measured by NanoDrop2000 (Thermo scientific) and the quality was checked by denaturing agarose gel electrophoresis. The cDNA synthesis was performed with reverse transcription kits (RR047A, Perfect Real Time, Takara). For quantification of miR-487b-3p expression, miRNA specific complementary DNA was generated using miRNA stem-loop-specific primers (Table 1). The cDNA generated was stored at -20°C for subsequent usage.
RT-qPCR was performed using TB green premix Ex taq II (RR820A, Perfect Real Time, Takara) on a Light Cycler 96 real-time system (Roche) with a 25 μL reaction volume. Each sample was carried out in triplicate and was repeated for three times at least. The mRNA expression levels of all coding genes were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal standard. On the other hand, the expression level of miR-487b was quantified with 18S RNA as the reference. The relative gene expression was analyzed using the comparative Ct (2−△△Ct) method.
Construction of CRISPR/Cas9 and RPG surrogate reporter vectors
In order to knockout miR-487b, three top single guide RNAs (sgRNAs) were designed according to the genomic sequence of Capra hircus miR-487b-3p (chi-miR-487b-3p) using the CRISPR Design Tool (http://chopchop.cbu.uib.no/) based on the following criteria: the sgRNA should have a high efficiency score and low off-target effects. Single-strand DNA annealing oligonucleotides for the guide and the target sequences of the three sgRNAs were synthesized by Invitrogen (Shanghai, China, as shown in table 2). Then, Annealed oligonucleotides for the three sgRNA guides were cloned into the Bbs I sites of the plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) (plasmid #42230, Addgene, Cambridge, MA) to construct the corresponding sgRNA/Cas9 expression vectors. The SSA-RPG surrogate reporter vectors was designed and constructed as we previously reported [24]. Generally, the oligo-nucleotide annealed products with PAMs for the three sgRNA targets were inserted into the Not I/BamH I sites of the SSA-RPG parental vector to generate the corresponding surrogate reporter vectors, respectively. All the plasmid vectors were confirmed by sequencing.
Cell transfection, screening and T7E1 cleavage assays
Primary goat myoblast cells (1×106 cells/well) were seeded into 6-well plates and cultured in normal culture medium to reach 80% of confluence. The myoblast cells were transiently transfected with 3 μg plasmid DNA (2 μg sgRNA/Cas9 expression vector and 1 μg RPG surrogate reporter vector) using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA) following to the manufacturer’s protocol. The parental vector pX330 without sgRNA guide sequence served as a negative control. The medium was changed 4 hours after transfection and the cells were screened for another 48 hours with 1.0 μg/mL of puromycin (P8833, Sigma) supplemented. Surviving cells were expanded and the genomic DNAs were extracted using the E.Z.N.A.DNA Kit (OMEGA Bio-Tek) for PCR and T7E1 cleavage assay. PCR was conducted using the 2×Taq Master Mix (Novoprotein). The DNA fragments spanning target sites were amplified with the primers: F 5΄-GACCCAGTCCACATAC AGCAAG-3΄, R 5΄-CGATAATCGCATCACCATTAC-3΄. Purified PCR products were subject to T7E1-cleavage assay (M0302L, NEB, Ipswich, MA, USA), and the digested DNA fragments were detected by gel electrophoresis. The band intensity for different DNA fragments was measured by Image J software (Image Lab, http://imagej.net) and was used for calculating the indel frequencies within the target sites as reported previously [27]. The purified PCR products were also inserted into the pMD™19-T Vector (Takara) by TA cloning for sequencing verification.
In addition, for chemically synthesized siRNAs, cells were transfected at 50% density with 50 nM RNA using lipofectamine 3000. The siRNA sequences are shown in Supplementary table S1.
Myoblast cells clone detection and off-target effect analysis
After puromycin resistant screening, selected myoblast cells were seeded into a 96-well plate for single cell cloning. After 10-15 days further culture, the cells were collected with half of each cell clones were seeded into new 48-well plates and the remaining half for genomic DNA extraction. Then, the precursor sequence of miR-487b-3p were amplified by PCR for validating the genomic modification by sequencing.
To assess the off-target effects, the potential off-target sites (POTs) for each sgRNA were predicted according to an online design tool (http://crispr.mit.edu/) and Cas-OFFinder (http://www.rgenome.net/cas-offinder/). The genomic sequences of the POTs were also amplified by PCR for sequencing verification. The primer sequences are shown in Supplementary table S2.
Cell counting kit-8 (CCK-8) and EdU imaging assays
The miR-487b-3p KO myoblast cells were seeded at a density of 1×103 cells per well in a 96 well plate in growth medium. Six independent biological replicates for each treatment were conducted. After the maintained for 24 hours, the cells were switched to the medium with 10% Cell Counting Kit-8 (CCK-8) reagent (EQ829, DOJINDO) and were incubated for another 4 hours at 37℃, which was followed by the absorbance measurement at 450 nm using a SYNERGY/H1 microplate reader (BioTek). The intensity of the color was directly proportional to the number of viable cells in the sample.
In addition, EdU imaging assay was carried out using the 488 EdU Click Proliferation kit (Beyotime) according to the manufacturer’s instruction. Myoblast cells were incubated with EdU (50 mM) (Beyotime) in the culture medium for 2 hours. Following incorporation of EdU and fixation, myoblast cells were subjected to Click-iT reaction (Invitrogen) for 30 minutes in the dark to add biotin to the EdU, and the cell nuclei were stained with Hoechst 33342 for 10 minutes. Afterward, the cells were visualized by using a Cell imaging multifunctional detection system (BioTek) and the data were analyzed with Image J software.
Flow cytometric analysis
The miR-487b-3p KO myoblast cells were seeded in 6-well plates at a density of 5×105 cells per well. After 48-hour culture, cells were washed three times with PBS and harvested. The cells were collected and fixed in cold 70% ethanol at 4 °C overnight. After RNase A treatment (1 mg/mL) at 37℃ for 30 minutes, and then staining with 4,6-diamidino-2-phenylindole (DAPI) staining solution (Solarbio) (50 µg/mL DAPI, 0.2% Triton X-100, 100 µg/mL RNase, PBS) for 1 hour at 4℃ in the dark. Then the cell suspension was centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. Finally, the cells were re-suspended in 1 mL PBS and analyzed by a BD FACSAria™ III flow cytometry system (BD).
Dual-luciferase reporter assay
The 3'UTR of goat IRS1 mRNA were amplified from myoblast cells cDNA with the primers as shown in table 2. The wild type or mutant 3'UTR sequences of IRS1 were cloned into the psi-CHECK2 vector (Promega, Germany) with restriction sites of Xho I and Not I, respectively. HEK293T cells were seeded in 48-well culture plates at a density of 1×104 cells per well. The cells were co-transfected with 250 ng of the wild-type (WT, psi-CHECK2-IRS1-3'UTR) or the mutant (Mutant, psi-CHECK2-IRS1-3'UTR-mut) 3'UTR plasmids and 100 nM miR-487b-3p mimics or negative control (NC) using the Lipofectamine 3000 reagent (L3000015, Invitrogen, Waltham, MA) following the manufacturers protocol. After the transfection for 48 hours, cells were harvested and lysed in passive lysis buffer (Promega, USA), and the dual-luciferase activity assay was performed according to the manufacturer’s instruction (Promega, USA).
miRNA target gene prediction and analysis
The sequences of miRNAs were obtained from the miRNA Registry miRBase (http://www.mirbase.org/) and the 3'UTR sequence of IRS1 were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/). The target genes of miRNA were predicted by TargetScan (http://www.targetscan.org), miRDB (http://www.mirdb.org/miRDB/) and David Bioinformatics analysis (https:// david.ncifcrf.gov/).
Protein extraction and western blot analysis
The total protein was extracted using RIPA lysis buffer (R0010, Solarbio, Beijing, China) with a protease inhibitor mix (04693132001, Roche Diagnostics, Ltd., Mannheim, Germany) after washing the cells with PBS three times. The protein concentration was determined using BCA Protein Assay kit (23227, Thermo Fisher Scientific, Rockford, IL). 30 μg of protein for each sample was separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) followed by transferring onto PVDF (polyvinylidene fluoride) membrane (HATF00010, Millipore, Burlington, MA). The membrane was blocked with 5% defatted milk (232100, BD, Franklin Lakes, NJ) in TBST (Tris-buffered saline with Tween 20) buffer for 1 hours at room temperature, and then was incubated with antibodies (1:1000) against Proliferating cell nuclear antige (PCNA) (Abcam), Cyclin dependent kinases 2 (CDK2) (SAB), Cyclin E, p27 (Santa Cruz) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (diluted 1:2000) (Bioss) at 4℃ overnight. The following day each membrane was washed three times with TBST, for 10 minutes each time. Subsequently, the membrane was incubated with HRP-conjugated secondary antibodies (anti-mouse IgG or anti-rabbits IgG) (Bioss) diluted at 1:2000. Chromogenic reaction was performed using an enhanced chemiluminescent (ECL) western blotting substrate substrate (K-12045-D10, Advansta, California, USA) and was detected by Sage Capture TM System (BioTek). Image J software was used for densitometric analysis.
Statistical analysis
All statistics were analyzed by GraphPad Prism 6.0 software and the data was expressed as the “mean ± SEM” of at least three independent repeats. The statistical significance of difference was assessed by unpaired Student’s t-test for two group comparisons and a one-way ANOVA for more than two groups. The difference was considered significant when the corresponding P value was less than 0.05 (*) or 0.01 (**).