Ethics statements
All experiments were performed according to the ethical standards of the institute. Animal procedures including biopsy collection were approved by the Institute Animal Ethics Committee, ICAR-National Dairy Research Institute, Karnal, India. The study is reported in accordance with ARRIVE guidelines, and approved by Institutional Biosafety Committee (IBSC).
CRISPR single guide RNA (sgRNA) design and transfection of buffalo fibroblasts
To edit BLG in buffalo, we used CHOPCHOP software (https://chopchop.cbu.uib.no/) with BLG gene ID, default NGG PAM, and 20 nucleotide sgRNA length settings to design three sgRNAs targeting exon 2 and 3 of the BLG. The designed sgRNA candidates were in-vitro transcribed using precision gRNA synthesis kit (Thermo Scientific#A29377) as per the manufacturer’s instructions, and then co-transfected with Cas9 protein (Thermo Scientific#A36498) into newborn female buffalo fibroblasts that were established as per our lab protocol13. The cells at passage 2 were transfected using the Amaxa nucleofector reagents (Lonza, Basel, Switzerland), according to program EN150 as per the manufacturer’s guidelines. Transfected cells were cultured for 72 h, and then, collected for genomic DNA extraction and generation of single cell clonal lines. The T7E1 assay and Sanger sequencing were performed to verify status of targeted BLG gene editing.
Detection of editing rates
The genomic DNA from transfected cells was extracted utilizing the Wizard genomic DNA purification kit, (Promega#A1125) as per the manufacturer’s instructions. The BLG primers (for exon 2, F- TGCCCCTCAAATTTTCCCCA and R-AAAGCCCTGGATAAGCAGCC; and for exon 3, F-CTGGCCCTCAGTTCATCCT and R-AGCAAAGAGAGCTCGGGTGT) were designed using PRIMER3 software (http://bioinfo.ut.ee/primer3-0.4.0/) and homology checked through nblast against nucleotide database of NCBI. The target sequence was subsequently amplified through polymerase chain reaction (PCR) under the following conditions: initial denaturation at 94 °C for 5 min, followed by 40 cycles at 94 °C for 20 s, 60 °C for 30 s, 72 °C for 35 s, and a final extension at 72 °C for 5 min. The PCR products from each sample underwent assessment using the T7E1 assay to detect editing in the BLG gene. Following denaturation and annealing of PCR products in NEB buffer 2 using a thermocycler, the hybridized PCR products underwent digestion with T7 endonuclease 1 (NEB#M0302L) for a duration of 20 min at 37 °C. The digested products were then subjected to 2% agarose gel electrophoresis. The gel images were analysed using the Gel Analyzer 19.1 software (www.gelanalyzer.com). The editing rate was determined by employing the formula: efficiency = [(sum of cleaved band intensities/(sum of cleaved and parental band intensities)] × 100. For TIDE and ICE analysis, PCR products were submitted for Sanger sequencing and resulted files were uploaded to the TIDE web tool (http://tide.nki.nl) and the ICE web tool (https://ice.synthego.com) for analysis. Both algorithms provided the percentage of insertions and deletions (indels).
Establishment of single cell clones and their genotype analysis
After 72 h of transfection, cells were trypsinized and the cell pellet was suspended in culture medium. The resuspended cells were then dispersed at low density in 3 mL of culture medium in a 35-mm culture dish. Single cells were manually picked using a fine-pulled glass capillary under optical control with a stereo zoom microscope and transferred into individual wells of a 96-well plate (one cell per well). The culture medium was supplemented with 10 ng/mL epidermal growth factor (Gibco#PHG0311) to enhance proliferation, and the EGF-supplemented culture media were refreshed every fifth day. Regular examination of cell clones was conducted using an inverted microscope, and morphology images were captured. Subsequently, cells from each clone were expanded, analyzed, and cryopreserved for future use. In the established single cell clones, BLG editing was assessed by analyzing targeted BLG-specific PCR products. These PCR products from each single cell clone were ligated into the pJet1.2/blunt TA cloning vector using the Clone JET PCR cloning kit (Thermo Scientific#K1232). Following this, the cloned PCR products were transformed into E.coli cells, and plasmids were isolated from 8 to 10 E. coli colonies using a geneJET plasmid miniprep kit (Thermo Scientific#K0503). The isolated plasmids underwent the Sanger sequencing, and the obtained data were analyzed to determine the actual editing genotypes.
Off-target analysis
We computationally predicted potential off-targets (OTs) of sgRNA2 using Cas-Offinder (http://www.rgenome.net/cas-offinder/), aligning with the buffalo genome assembly (NDDB_SH_1). Five OTs containing NGG PAM were selected (supplementary table 1 and 2). Off-target sites were amplified via conventional PCR, followed by Sanger sequencing. Analysis were done on three single cell clones, namely C2, C10, and C13, to detect any potential off-target effects.
Protein structure prediction from edited sequences
Firstly, we retrieved the nucleotide sequence (Accession no. AJ005429.1) and the corresponding amino acid sequence (Accession no. CAA06532.1) of the wild type BLG from the NCBI database. We targeted exon 2 of the BLG, and edited DNA sequences were placed into the corresponding wild type nucleotide sequences to generate the complete coding sequences. Subsequently, we utilized the ExPASy translation tool (https://web.expasy.org/translate/) to predict the open reading frames (ORFs) of the edited nucleotide sequences. To identify suitable ORF templates for further analysis, we performed the BLASTp, and compared the edited ORFs and the wild-type BLG protein (P02755, available at the UniProt database). This step aimed to select the appropriate templates for 3D structure prediction. Multiple alignments of the predicted amino acid sequences and the wild-type BLG were carried out using T-coffee (https://www.ebi.ac.uk/Tools/msa/tcoffee/). The protein structure prediction relies on the alignment between the wild type BLG sequence and edited ORF templates. For the actual 3D structure of the predicted amino acid sequences, we employed AlphaFold2, a highly efficient deep learning and artificial intelligence based tool (https://alphafold.ebi.ac.uk/). The resulting predicted structures were visualized using the PyMOL molecular graphics system, Version 2.0, by Schrӧdinger LLC (https://pymol.org/2/).
Production of cloned embryos
The handmade cloning (HMC) method, which was reported by us14, was employed for the production of BLG-edited cloned embryos. Briefly, buffalo ovaries were collected from a local abattoir and transported to the laboratory in normal saline within 4–6 h, and oocytes were harvested. In-vitro matured oocytes were denuded and subjected to zona pellucida removal, followed by manual bisection of protrusion cone-bearing oocytes. For nuclear transfer, donor cells, comprising BLG-edited fibroblasts, were paired with enucleated cytoplasts using phytohemagglutinin. The wild type fibroblasts were used as control. The resulting couplets underwent a single step electrofusion to form the triplets composed of a demi-oocyte, somatic cell, and another demi-oocyte. After 4 h incubation, the reconstructed oocytes were activated with calcimycin A23187, followed by 6-dimethylaminopurine treatment. The activated embryos were cultured in Research Vitro Cleave medium (K-RVCL-50, Cook®, Australia) supplemented with 1% fatty acid-free BSA in 4-well dishes, with 15–20 embryos per well. The dishes were covered with mineral oil and kept undisturbed in a CO2 incubator for 8 days. On day 8, blastocyst production rates were evaluated as indicators of in-vitro developmental competence.
Evaluation of embryo quality
For the evaluation of embryo quality, day 8 blastocysts were assessed for the total cell count and apoptosis index through TUNEL staining. Blastocysts were washed thrice with Dulbecco’s phosphate-buffered saline (DPBS) containing 0.3% polyvinyl alcohol (PVA) in a 4-well dish and fixed in 4% paraformaldehyde for 1 h at 37 °C. After additional washes, the blastocysts were stored at 4 °C until staining. Permeabilization was achieved by incubating blastocysts with 0.5% triton X-100 for 40 min, followed by incubation with FITC-conjugated dUTP and terminal deoxynucleotidyltransferase (TdT) for 90 min at 37 °C in the dark. Subsequently, the embryos were treated with nuclear staining solution (10 µg/mL Hoechst 33342 and 50 µg/mL RNase in DPBS + 0.3% PVA) for 25 min at 37 °C. Stained blastocysts were mounted on glass slides, and images were captured using both UV and green filters to examine nuclei and apoptosis sites, respectively. Digital images obtained from an inverted fluorescence microscope were used for analysis. The apoptotic index for each blastocyst was calculated as follows: Apoptotic index of blastocyst = (number of TUNEL-positive nuclei/total number of nuclei in blastocyst) × 100.
Statistical analysis
Statistical analysis was conducted using GraphPad Prism 5 software. The datasets underwent analysis through one-way analysis of variance (ANOVA), followed by the Tukey test for post hoc comparisons. Percentage values were subjected to arcsine transformation before analysis. Statistical significance was considered at a threshold of P < 0.05. The results are presented as mean ± standard error of the mean (SEM).
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- Source: https://www.nature.com/articles/s41598-024-65359-9