CRISPR-Cas9-generated mouse model of neurofibromatosis type 1

다국어 초록 (Multilingual Abstract)

Background To date, no experiments have been conducted to generate a neurofi bromatosis 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 modifi cation of mouse embryonic fi broblast NIH3T3 cells and embryos. The insertion– deletion (indel) effi cacy 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 effi cacy and pattern of NF1. Two of the four candidates with a 50% indel effi cacy were selected for embryo editing; however, without an appropriate sgRNA concentration, biallelic mutants were generated in 60–80% of the cases. Thus, by fi nding 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 confi rmed using NGS.
Conclusion Our study showed that the CRISPR-Cas9 embryo editing method is an effi cient tool for creating NF1 heterozygous ( NF1 +/− ) animal model.

참고문헌 (Reference)

• 1 Hur JK, "Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins" 34 : 807-808, 2016
• 2 Guo X, "Targeted genome editing in primate embryos" 25 : 767-768, 2015
• 3 Wang H, "One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering" 153 : 910-918, 2013
• 4 Rad E, "Neurofibromatosis type 1 : Fundamental insights into cell signalling and cancer" 52 : 39-46, 2016
• 5 Gutmann DH, "Neurofibromatosis type 1" 3 : 17004-, 2017
• 6 Leier A, "Mutation-directed therapeutics for neurofibromatosis type I" 20 : 739-753, 2020
• 7 Gutmann DH, "Mouse models of neurofibromatosis 1 and 2" 4 : 279-290, 2002
• 8 Gottfried ON, "Molecular, genetic, and cellular pathogenesis of neurofibromas and surgical implications" 58 : 1-16, 2006
• 9 Li K, "Mice with missense and nonsense NF1 mutations display divergent phenotypes compared with human neurofibromatosis type I" 9 : 759-767, 2016
• 10 Paquet D, "Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9" 533 : 125-129, 2016
• 11 Tanihara F, "Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos" 65 : 209-214, 2019
• 12 Taegeun Bae ; Woo Chang Hwang ; Dohyeon Lee ; 송길태 ; Junseok W Hur ; 허준호, "Characterization of distinct mutation patterns by CRISPR-Cas9 and CRISPR-Cpf1 genome editing systems" 대한독성 유전단백체 학회 15 (15): 383-389, 2019
• 13 Oji A, "CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice" 6 : 31666-, 2016