Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell–derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

Introduction
 Materials and Methods
  Primary culture of porcine fetal fibroblasts
  Generation of klotho knockout cell lines
  T7 endonuclease I assay
  Deep sequencing
  Somatic cell nuclear transfer
 Results and Discussion
  Klotho knockout in porcine fibroblasts via delivery of CRISPR/Cas9RNPs
  SCNT using a homozygous klotho knockout cell line
 REFERENCES

• Sanghoon Lee(Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea)
• Min Hee Jung(Toolgen, Inc., Seoul, Korea)
• Hyun Ju Oh(Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea)
• Ok Jae Koo(Toolgen, Inc., Seoul, Korea)
• Se Chang Park(Laboratory of Aquatic Biomedicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul, Korea)
• Byeong Chun Lee(Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea) Correspondence