T7E1 cleavage assay and Sanger sequencing of the PCR amplicons

Ear genomic DNA was extracted using a DNA extraction kit (Tiangen, DP348), and genomic DNA was extracted from rabbit embryos and amplified using the REPL1-g Single Cell Kit (Qiagen, 150343) following the manufacturer’s protocol. The genomic regions surrounding each CRISPR target site were amplified using PCR using primers listed in Table S4, then purified using QiaQuick Spin Column (Qiagen, 28704) following manufacturer’s protocol. A total of 200 ng of the purified PCR product was mixed with NEB Buffer 2, and subjected to the reannealing process to enable heteroduplex formation: 95 °C for 10 min, 95 °C to 85 °C ramping at −2 °C/s, 85 °C to 25 °C at −0.25 °C /s, and 25 °C hold for 1 min. After reannealing, products were treated with 0.5 μl T7E1 (NEB, M0302L) for 30 min, and resolved on 2% agarose gel. The PCR products with mutations detected by T7E1 cleavage assay or Sanger sequencing were then sub-cloned into pMD-19T vector (Takara, D103A). For each sample, 8 to 25 random colonies were used for sequencing.