Influence of homology arm length and structure on the efficiency of long transgene integration into a cleavage site induced by SpCas9 or AsCpf1
- Authors: Taran J.А.1, Mintaev R.R.1, Glazkova D.V.1, Belugin B.V.1, Bogoslovskaya E.V.1, Shipulin G.A.1
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Affiliations:
- Centre for Strategic Planning, of the Federal Medical and Biological Agency
- Issue: Vol 59, No 2 (2025)
- Pages: 255-265
- Section: МОЛЕКУЛЯРНАЯ БИОЛОГИЯ КЛЕТКИ
- URL: https://kazanmedjournal.ru/0026-8984/article/view/682880
- DOI: https://doi.org/10.31857/S0026898425020079
- EDN: https://elibrary.ru/GGBDOW
- ID: 682880
Cite item
Abstract
One of the promising new approaches to HIV infection treatment is the CRISPR/Cas-mediated knockout of the CCR5 receptor gene followed by the integration of an anti-HIV gene into the break site. Numerous studies have focused on the knockout of the CCR5 gene; however, the efficiency of subsequent targeted integration of long fragments remains poorly studied. To evaluate the efficiency of this approach, we used HT1080 cells and investigated the integration of a cassette expressing the EGFP gene into the CCR5 locus using two different nucleases (SpCas9 and AsCpf1) and various donor DNA constructs delivered by recombinant adeno-associated viral vectors (rAAV). For each nuclease, we designed five variants of donor DNA differing in the length (ranging from 150 to 1000 bp) or structure of the homology arms. The efficiency of transgene integration with 150 bp homology arms was the lowest for both nucleases and significantly differed from constructs with longer homology arms. Furthermore, it was shown that the presence of nuclease cleavage sites in the donor DNA flanking the cassette with homology arms did not affect the efficiency of transgene integration during AAV delivery. We demonstrated that the AsCpf1 nuclease provided higher efficiency of EGFP transgene integration than SpCas9, despite lower efficiency of CCR5 knockout. The maximum percentage of cells with integrated transgene was achieved using the AsCpf1 nuclease and an expression cassette with 600 bp homology arms, reaching 59 ± 6%.
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About the authors
J. А. Taran
Centre for Strategic Planning, of the Federal Medical and Biological Agency
Author for correspondence.
Email: taran.julia01@gmail.com
Russian Federation, Moscow
R. R. Mintaev
Centre for Strategic Planning, of the Federal Medical and Biological Agency
Email: taran.julia01@gmail.com
Russian Federation, Moscow
D. V. Glazkova
Centre for Strategic Planning, of the Federal Medical and Biological Agency
Email: taran.julia01@gmail.com
Russian Federation, Moscow
B. V. Belugin
Centre for Strategic Planning, of the Federal Medical and Biological Agency
Email: taran.julia01@gmail.com
Russian Federation, Moscow
E. V. Bogoslovskaya
Centre for Strategic Planning, of the Federal Medical and Biological Agency
Email: taran.julia01@gmail.com
Russian Federation, Moscow
G. A. Shipulin
Centre for Strategic Planning, of the Federal Medical and Biological Agency
Email: taran.julia01@gmail.com
Russian Federation, Moscow
References
- Mautino M.R. (2002) Lentiviral vectors for gene therapy of HIV-1 infection. Curr. Gene Ther. 2, 23–43.
- Orlova O.V., Glazkova D.V., Mintaev R.R., Tsyganova G.M., Urusov F.A., Shipulin G.A., Bogoslovskaya E.V. (2023) Comparative evaluation of the activity of various lentiviral vectors containing three anti-HIV genes. Microorganisms. 11, 1053.
- Chang L.-J., Liu X., He J. (2005) Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1. Gene Ther. 12, 1133–1144.
- Milone M.C., O’Doherty U. (2018) Clinical use of lentiviral vectors. Leukemia. 32, 1529–1541.
- Naeem M., Majeed S., Hoque M.Z., Ahmad I. (2020) Latest developed strategies to minimize the off-target effects in CRISPR-Cas-mediated genome editing. Cells. 9, 1608.
- Papapetrou E.P., Schambach A. (2016) Gene insertion into genomic safe harbors for human gene therapy. Mol. Ther. 24, 678–684.
- Lucotte G. (2002) Frequencies of 32 base pair deletion of the (Delta 32) allele of the CCR5 HIV-1 co-receptor gene in Caucasians: a comparative analysis. Infect. Genet. Evol. 1, 201–205.
- Karuppusamy K.V., Demosthenes J.P., Venkatesan V., Christopher A.C., Babu P., Azhagiri M.K., Jacob A., Ramalingam V.V., Rangaraj S., Murugesan M.K., Marepally S.K., Varghese G.M., Srivastava A., Kannangai R., Thangavel S. (2022) The CCR5 gene edited CD34+CD90+ hematopoietic stem cell population serves as an optimal graft source for HIV gene therapy. Front. Immunol. 13, 792684.
- Xu L., Yang H., Gao Y., Chen Z., Xie L., Liu Y., Liu Y., Wang X., Li H., Lai W., He Y., Yao A., Ma L., Shao Y., Zhang B., Wang C., Chen H., Deng H. (2017) CRISPR/Cas9-mediated CCR5 ablation in human hematopoietic stem/progenitor cells confers HIV-1 resistance in vivo. Mol. Ther. 25, 1782–1789.
- Liu M., Rehman S., Tang X., Gu K., Fan Q., Chen D., Ma W. (2019) Methodologies for improving HDR efficiency. Front. Genet. 9. 691. doi: 10.3389/fgene.2018.00691
- Zhang J.-P., Li X.L., Li G.H., Chen W., Arakaki C., Botimer G.D., Baylink D., Zhang L., Wen W., Fu Y.W., Xu J., Chun N., Yuan W., Cheng T., Zhang X.B. (2017) Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol. 18, 35.
- Tóth E., Weinhardt N., Bencsura P., Huszár K., Kulcsár P.I., Tálas A., Fodor E., Welker E. (2016) Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells. Biol. Direct. 11, 46.
- Irion U., Krauss J., Nüsslein-Volhard C. (2014) Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. Development (Cambridge, England). 141, 4827–4830.
- Yao X., Zhang M., Wang X., Ying W., Hu X., Dai P., Meng F., Shi L., Sun Y., Yao N., Zhong W., Li Y., Wu K., Li W., Chen Z.J., Yang H. (2018) Tild-CRISPR allows for efficient and precise gene knockin in mouse and human cells. Dev. Cell. 45, 526–536.e5.
- Bak R.O., Porteus M.H. (2017) CRISPR-mediated integration of large gene cassettes using AAV donor vectors. Cell Rep. 20, 750–756.
- Глазкова Д.В., Ветчинова А.С., Богословская Е.В., Жогина Ю.А., Маркелов М.Л., Шипулин Г.А. (2013) Подавление экспрессии гена CCR5-рецептора человека с помощью искусственных микроРНК. Молекуляр. биология 47(3), 475–485.
- Kimura T. Ferran B., Tsukahara Y., Shang Q., Desai S., Fedoce A., Pimentel D.R., Luptak I., Adachi T., Ido Y., Matsui R., Bachschmid M.M. (2019) Production of adeno-associated virus vectors for in vitro and in vivo applications. Sci. Rep. 9, 13601.
- Guo P., El-Gohary Y., Prasadan K., Shiota C., Xiao X., Wiersch J., Paredes J., Tulachan S., Gittes G.K. (2012) Rapid and simplified purification of recombinant adeno-associated virus. J. Virol. Methods. 183, 139–146.
- Минтаев Р.Р., Глазкова Д.В., Таран Ю.А., Богословская Е.В., Шипулин Г.А. (2025) Повышение эффективности и безопасности редактирования гена CCR5 человека путем подбора оптимальных направляющих РНК Cas9 и Cas12a. Молекуляр. биология. 59(2), ХХ-ХХ.
- Gutierrez-Guerrero A., Abrey Recalde M.J., Mangeot P.E., Costa C., Bernadin O., Périan S., Fusil F., Froment G., Martinez-Turtos A., Krug A., Martin F., Benabdellah K., Ricci E.P., Giovannozzi S., Gijsbers R., Ayuso E., Cosset F.L., Verhoeyen E. (2021) Baboon envelope pseudotyped “Nanoblades” carrying Cas9/gRNA complexes allow efficient genome editing in human T, B, and CD34+ cells and knock-in of AAV6-encoded donor DNA in CD34+ cells. Front. Genome Editing. 3, 604371.
- Safari F., Zare K., Negahdaripour M., Barekati-Mowahed M., Ghasemi Y. (2019) CRISPR Cpf1 proteins: structure, function and implications for genome editing. Cell Biosci. 9, 36.
- Maslennikova A., Mazurov D. (2022) Application of CRISPR/Cas genomic editing tools for HIV therapy: toward precise modifications and multilevel protection. Front. Cell. Infect. Microbiol. 12, 880030. doi.org: 10.3389/fcimb.2022.880030.
- Hung K.L., Meitlis I., Hale M., Chen C.Y., Singh S., Jackson S.W., Miao C.H., Khan I.F., Rawlings D.J., James R.G. (2018) Engineering protein-secreting plasma cells by homology-directed repair in primary human B cells. J. Am. Soc. Gene Ther. 26, 456–467.
- Hendel A., Kildebeck E.J., Fine E.J., Clark J., Punjya N., Sebastiano V., Bao G., Porteus M.H. (2014) Quantifying genome editing outcomes at endogenous loci using SMRT sequencing. Cell Rep. 7, 293–305.
- Chu V.T., Weber T., Wefers B., Wurst W., Sander S., Rajewsky K., Kühn R. (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548.
- Odé Z., Condori J., Peterson N., Zhou S., Krenciute G. (2020) CRISPR-mediated non-viral site-specific gene integration and expression in T cells: protocol and application for T-cell therapy. Cancers. 12, 1704.
- Shin S.W., Lee J.S. (2020) Optimized CRISPR/Cas9 strategy for homology-directed multiple targeted integration of transgenes in CHO cells. Biotechnol. Bioeng. 117, 1895–1903.
- Kim S., Kim D., Cho S.W., Kim J., Kim J. S. (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019.
- Asaad W., Volos P., Maksimov D., Khavina E., Deviatkin A., Mityaeva O., Volchkov P. (2023) AAV genome modification for efficient AAV production. Heliyon. 9, e15071.
- Zetsche B., Gootenberg J.S., Abudayyeh O.O., Slaymaker I.M., Makarova K.S., Essletzbichler P., Volz S.E., Joung J., van der Oost J., Regev A., Koonin E.V., Zhang F. (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 163, 759–771.
- Ding R., Chao C.-C., Gao Q. (2022) High-efficiency of genetic modification using CRISPR/Cpf1 system for engineered CAR-T cell therapy. Methods Cell Biol. 167, 1–14.
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