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KMID : 1034820230190020277
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Molecular & Cellular Toxicology
2023 Volume.19 No. 2 p.277 ~ p.282
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CRISPR-Cas9-generated mouse model of neurofibromatosis type 1
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Park Tae-Gun
Ye Sung-Hyeok
Shin Sang-Kyu
Kim Kyoung-Mi
Junho K. Hur
Junseok W. Hur
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Abstract
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Background : To date, no experiments have been conducted to generate a neurofibromatosis type 1 (NF1) mouse model using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) embryo editing system.
Objective : This study was to deliver ribonucleoprotein (RnP) via electroporation in various formats and delivery methods of the CRISPR-Cas9 system for genetic modification of mouse embryonic fibroblast NIH3T3 cells and embryos. The insertion?deletion (indel) efficacy and pattern of NF1 were analyzed using next-generation sequencing (NGS).
Results : We established four candidate single-guide RNAs (sgRNAs) and transfected them with Streptococcus pyogenes Cas9 (SpCas9) protein (RnP) via electroporation into NIH3T3 cells to analyze the indel efficacy and pattern of NF1. Two of the four candidates with a 50% indel efficacy were selected for embryo editing; however, without an appropriate sgRNA concentration, biallelic mutants were generated in 60?80% of the cases. Thus, by finding an appropriate concentration for RnP, it was possible to increase the rate of monoallelic mutant generation. Finally, we successfully produced an NF1 heterozygote mouse model, and the mutant sequence was confirmed using NGS.
Conclusion : Our study showed that the CRISPR-Cas9 embryo editing method is an efficient tool for creating NF1 heterozygous (NF1+/?) animal model.
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KEYWORD
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Neurofibromatosis type 1, NF1, CRISPR-Cas9, Gene editing, Mouse model, Embryo editing
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