Snabi

Following linearization of SLC39A14-EGFP constructs with SnaBI (NEB), capped mRNA was generated using the mMessage mMachine Sp6 Transcription Kit (Life Technologies) and purified using the RNeasy Mini Kit (Qiagen). About 50 pg of either SLC39A14 mRNA (transcript 1/2) was co-injected with 50 pg mRNA encoding membrane mCherry. Injected embryos were fixed at 6 hpf in 4% paraformaldehyde at 4 °C overnight. Following several washes in PBS/0.1% Triton X-100 embryos were blocked in 10% goat serum at room temperature for 1 h. This was followed by incubation with primary antibody (chicken anti-GFP (1:500, ab13970, Abcam), rabbit anti-RFP (1:1,000; PM005, MBL)) at 4 °C overnight. Following washes in PBS/0.1% Triton X-100, embryos were incubated in fluorescent secondary antibody (goat anti-chicken Alexa Fluor 488 (1:200, A-11039, Life Technologies), goat anti-rabbit Alexa Fluor 568 (1:200, A-11011, Life Technologies)) at 4 °C overnight. DAPI (1:500, Life Technologies) was used for nuclear staining. Embryos were mounted in 1.5% low melt agarose and imaged on a Leica TCS SPE confocal microscope using a × 25 0.95 water-immersion objective. Z-stacks were acquired in 1-μm intervals and maximum-intensity projections generated with Fiji software62 (link).

DNA oligonucleotides (Supplementary Table 12 and Supplementary Fig. 21) were purchased from IDT (Leuven, Belgium) and HPLC purified. The DNA substrates were obtained by folding or hybridization in 20 mM MOPS pH 7.4, 75 mM NaCl, 2 mM MgCl₂, at 85 °C for 3 min followed by slow cooling to room temperature before storage at −20 °C. E.coli genomic DNA (Sigma) was digested with EcoRV and SnaBI (NEB) at 37 °C for 4 h to create blunt-end DNA substrates. DNA concentration was calculated by measuring absorbance at 260 nm and the number of moles of dsDNA was calculated according to E.coli genomic restriction map analysis.

Putative KI embryos prepared by ldsDNA electroporation or AAV infection were cultured for 5 days in mR1ECM to develop them into 8-cell embryos to blastocysts. The zona pellucida was removed using acidic Tyrode’s solution (Sigma-Aldrich), and then directly subjected to genomic PCR using Tks Gflex DNA polymerase and the primers listed in Table S1. PCR fragments were electrophoresed and sequenced for further confirmation. For PCR-RFLP analysis, Tyr alleles were amplified by PCR and digested using the restriction enzyme SnaBI (New England Biolabs), and electrophoresed to detect the KI allele.

A total of 1×10⁶ cells were collected and crosslinked with 1% formaldehyde. After stopping the crosslinking via glycine, the cells were lysed using lysis buffer and centrifuged to remove cellular debris. The chromatin was then diluted 3-fold using a ChIP dilution buffer containing a protease inhibitor cocktail (Sigma-Aldrich) and digested overnight at 37°C with restriction enzymes including EcoNI, SnaBI, SalI, and NotI (New England Biolabs). The digested chromatin was further diluted 6-fold into a T4 ligation buffer before ligation was performed for 4 h at room temperature using T4 DNA ligase and 0.5 mM ATP. DNA was purified with the QIAquick PCR Purification Kit (Qiagen) followed by CHIP-qPCR. For CHIP assay, anti-DNMT3 (Abcam) was used to bind target protein-DNA complexes. The primers used in the 3C-ChIP-PCR assays are listed in the Table 2.

To obtain the fragment coding for the M41-S1 gene, RNA was isolated from a virus stock of IBV-M41 (Animal Health Service, The Netherlands) using the QIAamp viral RNA Mini Kit (Qiagen, Germany). Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland) with random hexamers according to the manufacturer’s protocol. PCR was performed with Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) using the primers listed in Table 1. To exchange the S1 domain of IBV-H52 for that of M41 in the plasmid used for targeted recombination, the M41-S1 PCR product and the previously generated plasmid containing the H52-S gene [16 (link)] were digested using restriction enzymes PacI and SnaBI (both New England Biolabs, USA), ligated and subsequently transformed in HB101 E. coli. Sequences were confirmed by automated nucleotide sequencing (Macrogen, The Netherlands). A step-wise ligation approach was used to obtain the H52 M41-S1 donor plasmid. To introduce mutations leading to individual N-to-A substitutions in N-glycosylation sites in the receptor binding domain (RBD) sequence, site-directed mutagenesis was performed using the primers listed in Table 1. The sequences of the plasmids containing the S1 gene with the introduced mutations were confirmed by automated nucleotide sequencing (Macrogen, The Netherlands).

Restriction enzymes SnabI and SalI (New England Biolabs, Ipswich, MA) were used to digest the human catalase sequence from the CAT-pC1 plasmid [36 (link)], which was then subcloned into the pBABE-puro retroviral vectors (a gift from Hartmut Land and Jay Morgenstern and Bob Weinberg, Addgene plasmid # 1764). After verification by sequencing, these retroviral constructs were co-transfected into HEK293T cells with two packaging vectors VSV-G, and Gag-Pol using Fugene 6 (Promega, E2692, Madison, WI). Two days after transfection, the culture supernatant containing viruses was purified by filtration through 0.45 μm filters and stored at -80 °C. Puromycin selection was performed after virus transduction at 1 μg/ml in culture media for 3 days.