Study of the impact of nuclease Cas9 delivery methods on knockout efficiency in T lymphocytes using the CRISPR /Cas9 genome editing system in the experimental T cell line model Jurkat

Authors

  • Kathrine S. Pokladok Republican Scientific and Practical Center for Children’s Oncology, Hematology and Immunology, 43 Frunzienskaja Street, Barawliany 223053, Barawlianski sielsaviet, Minsk District, Belarus
  • Alexander N. Meleshko Republican Scientific and Practical Center for Children’s Oncology, Hematology and Immunology, 43 Frunzienskaja Street, Barawliany 223053, Barawlianski sielsaviet, Minsk District, Belarus
  • Maryia S. Kisel Institute of Bioоrganic Chemistry, National Academy of Sciences of Belarus, 5 Akademika Kuprevicha Street, 2 building, Minsk 220084, Belarus

Keywords:

CRISPR/Cas9, sgRNA, gene TRAC, gene B2M, knockout, CAR

Abstract

The development of allogeneic CAR-T cells represents a promising direction in cancer immunotherapy, overcoming the limitations of autologous approaches. One of the key steps in creating such cells is the efficient delivery of CRISPR/Cas9 genome editing components into them. This article explores strategies for optimising electroporation parameters for nuclease Cas9 (mRNA, plasmid vector, protein) to enhance genome editing efficiency. The optimised protocol may contribute to the standardisation of allogeneic CAR-T cell manufacturing, improving their therapeutic efficacy and safety.

Author Biographies

  • Kathrine S. Pokladok, Republican Scientific and Practical Center for Children’s Oncology, Hematology and Immunology, 43 Frunzienskaja Street, Barawliany 223053, Barawlianski sielsaviet, Minsk District, Belarus

    junior researcher at the laboratory of genetic biotechnologies

  • Alexander N. Meleshko, Republican Scientific and Practical Center for Children’s Oncology, Hematology and Immunology, 43 Frunzienskaja Street, Barawliany 223053, Barawlianski sielsaviet, Minsk District, Belarus

    leading researcher at the laboratory of genetic biotechnologies

  • Maryia S. Kisel, Institute of Bioоrganic Chemistry, National Academy of Sciences of Belarus, 5 Akademika Kuprevicha Street, 2 building, Minsk 220084, Belarus

    researcher at the laboratory of molecular diagnostics and biotechnology

References

  1. Кушнерова ЕВ, Мигас АА, Клыч АВ, Ласюков ЕА, Мелешко АН. Нокаут генов Т-клеточного рецептора и HLA класса I в клетках человека с использованием системы CRISPR /Cas9. Экспериментальная биология и биотехнология. 2022;2:19–26. DOI: 10.33581/2957-5060-2022-2-19-26.
  2. Покладок ЕС, Мелешко АН, Кушнерова ЕВ. Оценка эффективности нокаута Т-клеточного рецептора и MHCI в клетках Т-лимфоцитов с использованием различных экспрессионных кассет. В: Мартинович Г, Волотовский ИД, Лукьяненко ЛМ, Слобожанина ЕИ, Кабашникова ЛФ, Вересов ВГ и др., редакторы. Молекулярные, мембранные и клеточные основы функционирования биосистем. Тезисы докладов 16-й Международной научной конференции; 25–27 июня 2024 г.; Минск, Беларусь. Минск: БГУ; 2024. с. 263–264.
  3. Kagoya Y, Guo T, Yeung B, Saso K, Anczurowski M, Wang C-H, et al. Genetic ablation of HLA class I, class II, and the T cell receptor enables allogeneic T cells to be used for adoptive T cell therapy. Cancer Immunology Research. 2020;8(7):926–936. DOI: 10.1158/2326-6066.CIR-18-0508.
  4. Ren J, Liu X, Fang C, Jiang S, June CH, Zhao Y. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clinical Cancer Research. 2017;23(9):2255–2266. DOI: 10.1158/1078-0432.CCR-16-1300.
  5. Stenger D, Stief TA, Kaeuferle T, Willier S, Rataj F, Schober K, et al. Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR. Blood. 2020;136(12):1407–1418. DOI: 10.1182/blood.2020005185.
  6. Nakazawa Y, Huye LE, Dotti G, Foster AE, Vera JF, Manuri PR, et al. Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes. Journal of Immunotherapy. 2009;32(8):826–836. DOI: 10.1097/CJI.0b013e3181ad762b.

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Published

2026-02-17

Issue

No. 3 (2025): Experimental Biology and Biotechnology

Section

Genetics and Molecular Biology

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Copyright (c) 2026 Experimental Biology and Biotechnology

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How to Cite

Pokladok, K. S., Meleshko, A. N., & Kisel, M. S. (2026). Study of the impact of nuclease Cas9 delivery methods on knockout efficiency in T lymphocytes using the CRISPR /Cas9 genome editing system in the experimental T cell line model Jurkat. Experimental Biology and Biotechnology, 3, 38-47. https://doi.org/10.33581/2957-5060-2025-3-%p