M.N. Karagyaur1, A.L. Primak2, A.E. Tolstoluzhinskaya3, A.V. Reshetnev4, N.A. Basalova5, S.S. Dzhauari6, K.D. Bozov7, A.N. Velikanov8, A.Yu. Efimenko9, E.V. Semina10, L.M. Samokhodskaya11, P.S. Klimovich12, V.S. Popov13, D.A. Sheleg14, E.A. Neyfeld15

1–15 Medical Research and Education Institute Lomonosov Moscow State University (Moscow, Russia)

1 m.karagyaur@mail.ru, 2 primak.msu@mail.ru, 3 a.luzh@yandex.ru, 4 sasharesh@gmail.com, 5 natalia_ba@mail.ru, 6 stalik.djauari@yandex.ru, 7 kir-bozov@yandex.ru, 8 av-bioem@mail.ru, 9 efimenkoan@gmail.com, 10 e-tal@yandex.ru, 11 samokhodskay@gmail.com, 12 lex2050@mail.ru, 13 galiantus@gmail.com, 14 sheleg-da@mail.ru, 15 ea.neyfeld@mail.ru

The great diversity of technologies of cell culture genetic modification necessitates a clear understanding of their potential and limitations. Identification of the most efficient and accurate of them will save time to researchers in obtaining the desired cellular models.

Purpose of work – to conduct a comparative analysis of the potential of prime-editing, base editing and trans-splicing technologies for modeling of sought variants of human genes: ACTA2, ADRB2, ADRB3 and mouse Plaur gene.

We tested the efficiency of trans-splicing technology in two cellular models: 1. lifetime labelling of inducible ACTA2 protein expression in primary human dermal fibroblasts differentiating into myofibroblasts (in vitro model of fibrosis); 2. modeling a Plaur gene variant lacking the ability to interact with vitronectin in Neuro2a neuroblastoma cell culture. No sought hybrid trans-spliced mRNAs were detected in lysates of myofibroblasts; however, the fact of non-target trans-splicing was revealed. Optimization of donor trans-RNAs didn’t increase the specificity and efficiency of trans-splicing. This technique is very sensitive to the design of the donor trans-RNAs, the optimization of which is time and resource consuming on a case-by-case basis. The efficiency of prime-editing technology was assessed using two different cellular models: conversion of fluorescence spectrum of HEK293T cells transfected with fluoPEER-GFP-BFP genetic construct and modeling of Q33Stop variant in Plaur gene in Neuro2a cell culture. Prime-editing provided relatively low efficiency of the desired modification with a much higher probability of generating non-target products. In contrast, base editing technology allows modifying at least 50% of the total number of alleles in the cell population, with minimal off-target activity, which has been confirmed by the results of Sanger sequencing and flow cytometry of the modified murine neuroblastoma cells and immortalized human mesenchymal stromal cells.

The data obtained do not allow us to consider trans-splicing and prime-editing technologies as effective and specific approaches to modeling the desired genomic variants. According to our data, the most efficient, convenient and adapted for the creation of genetically modified cellular models is the base editing technology, which allows us to achieve an acceptable level of editing even in a non-cloned cell population.

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