Realities in the Creation of Humanized Transgenic Personalized Animals for Biomedical Research: Transition from Plasmid to CRISPR/Cas9 Ribonucleoprotein Complex

Creation of humanized genetically modified animals for biomedical research is an important direction in the activity of the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency (FMBA) of Russia. Since 2013, the Center has obtained more than 20 lines of transgenic, knockout, and transgenically-knockout mouse biomodels using classical transgenesis and the plasmid version of the CRISPR/Cas9 technology. The main specialized lines involve humanized transgenic mouse biomodels carrying alleles HLA class I — HLA-В*, HLA-В**, HLA-В***, HLA-С*, HLA-B*07:02, HLA-С*07:02, HLA-A*02:01, HLA-В*** КО, HLA-A** КО, HLA-B**** КО, HLA-С** КО, as well as NAT1, NAT2, АСЕ2 hom, hACE2-Tom (HDRKI), SMN2 (S2), PrPSc, АСЕ2 hom / HLA-C**, β2m mus KO, PrP КО, STE24 КО, SMN (S6) КО, SMN2 КО, PrP КО / PrPSc, NAT1 KO, NAT2 KO. In this article, we review the genetic constructs and methods used to produce genetically modified animals. The article summarizes the long-term experience of using the plasmid variant and discusses the need to switch to the CRISPR/Cas9 ribonucleoprotein complex in the view of its high efficiency and specificity, high frequency of targeted modifications, the ability to use multiple gRNAs, and the insertion of a single copy of the HDR-DNA template (transgene). We also explore the possibility of replacing imported reagents and kits with domestic products.