Improving the efficiency and safety of human CCR5 gene editing by selection of optimal guide RNAs for SpCAS9 and CAS12A

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Abstract

Advances in CRISPR/Cas-mediated genome editing have opened up treatment alternatives for many human diseases, including HIV infection. Knockout of the CCR5 gene as a potential way to treat HIV infection has long been studied. Here we analyzed guide RNAs for SpCas9 and AsCas12a nucleases targeting CCR5 gene which had been previously studied and selected the most effective among them. We also designed novel guide RNAs for the same nucleases using bioinformatics resources. We compared the efficiency of target site cleavage for all selected gRNAs using three nucleases: wt SpCas9, SpCas9-HF1-plus, and AsCas12a, as well as their off- target activities. We demonstrated that among the tested guide RNAs two for SpCas9-HF1-plus and three for AsCas12a exhibited high cleavage activity, cutting CCR5 gene in 60–72% of cells, and had off-target activities below the limit of detection. Thus, these guide RNAs may be candidates for future development of gene therapies against HIV infection.

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About the authors

R. R. Mintaev

Center for Strategic Planning and Management of Medical and Biological Health Risks, Federal Medical-Biological Agency

Author for correspondence.
Email: ramil.mintaev@fbb.msu.ru
Russian Federation, Moscow

D. V. Glazkova

Center for Strategic Planning and Management of Medical and Biological Health Risks, Federal Medical-Biological Agency

Email: ramil.mintaev@fbb.msu.ru
Russian Federation, Moscow

J. А. Taran

Center for Strategic Planning and Management of Medical and Biological Health Risks, Federal Medical-Biological Agency

Email: ramil.mintaev@fbb.msu.ru
Russian Federation, Moscow

E. V. Bogoslovskaya

Center for Strategic Planning and Management of Medical and Biological Health Risks, Federal Medical-Biological Agency

Email: ramil.mintaev@fbb.msu.ru
Russian Federation, Moscow

G. A. Shipulin

Center for Strategic Planning and Management of Medical and Biological Health Risks, Federal Medical-Biological Agency

Email: ramil.mintaev@fbb.msu.ru
Russian Federation, Moscow

References

  1. Cornu T.I., Mussolino C., Müller M.C., Wehr C., Kern W.V., Cathomen T. (2021) HIV gene therapy: an update. Hum. Gene Ther. 32, 52–65.
  2. Allers K., Hütter G., Hofmann J., Loddenkemper C., Rieger K., Thiel E., Schneider T. (2011) Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation. Blood. 117(10), 2791–2799.
  3. Hütter G., Nowak D., Mossner M., Ganepola S., Müssig A., Allers K., Schneider T., Hofmann J., Kücherer C., Blau O., Blau I.W., Hofmann W.K., Thiel E. (2009) Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N. Engl. J. Med. 360(7), 692–698.
  4. Peterhoff D. (2023) New case of HIV cure: joined forces of haploidentical stem cells and HLA-mismatched cord blood. Signal Transduct. Target. Ther. 8(1), 241.
  5. Doudna J.A., Charpentier E. (2014) Genome editing. The new frontier of genome engineering with CRISPR/Cas9. Science. 346, 1258096.
  6. Cong L., Ran F.A., Cox D., Lin S., Barretto R., Habib N., Hsu P.D., Wu X., Jiang W., Marraffini L.A., Zhang F. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science. 339, 819–823.
  7. Cho S.W., Kim S., Kim J.M., Kim J.S. (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232.
  8. Kang H., Minder P., Park M.A., Mesquitta W.T., Torbett B.E., Slukvin I.I. (2015) CCR5 disruption in induced pluripotent stem cells using CRISPR/Cas9 provides selective resistance of immune cells to CCR5-tropic HIV-1 virus. Mol. Ther. Nucl. Acids. 4, e268.
  9. Nerys-Junior A., Braga-Dias L.P., Pezzuto P., Cotta-de-Almeida V., Tanuri A. (2018) Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR/Cas9 when targeting the human CCR5 gene. Genet. Mol. Biol. 41, 167–179.
  10. Cradick T.J., Fine E.J., Antico C.J., Bao G. (2013) CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucl. Acids Res. 41, 9584–9592.
  11. Fine E.J., Appleton C.M., White D.E., Brown M.T., Deshmukh H., Kemp M.L., Bao G. (2015) Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Sci. Rep. 5, 10777.
  12. Dabrowska M., Czubak K., Juzwa W., Krzyzosiak W.J., Olejniczak M., Kozlowski P. (2018) qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites. Nucl. Acids Res. 46, e101.
  13. Yu S., Yao Y., Xiao H., Li J., Liu Q., Yang Y., Adah D., Lu J., Zhao S., Qin L., Chen X. (2018) Simultaneous knockout of CXCR4 and CCR5 genes in CD4+ T сells via CRISPR/Cas9 confers resistance to both X4- and R5-tropic human immunodeficiency virus type 1 infection. Hum. Gene Ther. 29, 51–67.
  14. Liu X., Wang M., Qin Y., Shi X., Cong P., Chen Y., He Z. (2018) Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9. BMC Biotechnol. 18, 66.
  15. 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.
  16. Xu L., Wang J., Liu Y., Xie L., Su B., Mou D., Wang L., Liu T., Wang X., Zhang B., Zhao L., Hu L., Ning H., Zhang Y., Deng K., Liu L., Lu X., Zhang T., Xu J., Li C., Wu H., Deng H., Chen H. (2019) CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. N. Engl. J. Med. 381(12), 1240–1247.
  17. Li C., Guan X., Du T., Jin W., Wu B., Liu Y., Wang P., Hu B., Griffin G.E., Shattock R.J., Hu Q. (2015) Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9. J. Gen. Virol. 96, 2381–2393.
  18. Mandal P.K., Ferreira L.M., Collins R., Meissner T.B., Boutwell C.L., Friesen M., Vrbanac V., Garrison B.S., Stortchevoi A., Bryder D., Musunuru K., Brand H., Tager A.M., Allen T.M., Talkowski M.E., Rossi D.J., Cowan C.A. (2014) Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell. 15, 643–652.
  19. Ehrke-Schulz E., Schiwon M., Leitner T., Dávid S., Bergmann T., Liu J., Ehrhardt A. (2017) CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes. Sci. Rep. 7, 17113.
  20. Hosseini Rouzbahani N., Kaviani S., Vasei M., Soleimani M., Azadmanesh K., Nicknam M.H. (2019) Generation of CCR5-ablated human induced pluripotent stem cells as a therapeutic approach for immune-mediated diseases. Iran. J. Allergy Asthma Immunol. 18, 310–319.
  21. Vakulskas C.A., Dever D.P., Rettig G.R., Turk R., Jacobi A.M., Collingwood M.A., Bode N.M., McNeill M.S., Yan S., Camarena J., Lee C.M., Park S.H., Wiebking V., Bak R.O., Gomez-Ospina N., Pavel-Dinu M., Sun W., Bao G., Porteus M.H., Behlke M.A. (2018) A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216–1224.
  22. Gomez-Ospina N., Scharenberg S.G., Mostrel N., Bak R.O., Mantri S., Quadros R.M., Gurumurthy C.B., Lee C., Bao G., Suarez C.J., Khan S., Sawamoto K., Tomatsu S., Raj N., Attardi L.D., Aurelian L., Porteus M.H. (2019) Human genome-edited hematopoietic stem cells phenotypically correct mucopolysaccharidosis type I. Nat. Commun. 10, 4045.
  23. Scharenberg S.G., Poletto E., Lucot K.L., Colella P., Sheikali A., Montine T.J., Porteus M.H., Gomez-Ospina N. (2020) Engineering monocyte/macrophage-specific glucocerebrosidase expression in human hematopoietic stem cells using genome editing. Nat. Commun. 11, 3327.
  24. Liu Z., Chen S., Jin X., Wang Q., Yang K., Li C., Xiao Q., Hou P., Liu S., Wu S., Hou W., Xiong Y., Kong C., Zhao X., Wu L., Li C., Sun G., Guo D. (2017) Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR/Cas9 protects CD4+ T cells from HIV-1 infection. Cell Biosci. 7, 47.
  25. Ye L., Wang J., Beyer A.I., Teque F., Cradick T.J., Qi Z., Chang J.C., Bao G., Muench M.O., Yu J., Levy J.A., Kan Y.W. (2014) Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. Proc. Natl. Acad. Sci. USA. 111, 9591–9596.
  26. Li X., Bai Y., Cheng X., Kalds P.G.T., Sun B., Wu Y., Lv H., Xu K., Zhang Z. (2018) Efficient SSA-mediated precise genome editing using CRISPR/Cas9. FEBS J. 285, 3362–3375.
  27. Kang X., He W., Huang Y., Yu Q., Chen Y., Gao X., Sun X., Fan Y. (2016) Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. J. Assist. Reprod. Genet. 33, 581–588.
  28. Qi C., Li D., Jiang X., Jia X., Lu L., Wang Y., Sun J., Shao Y., Wei M. (2018) Inducing CCR5Δ32/Δ32 homozygotes in the human jurkat CD4+ cell line and primary CD4+ cells by CRISPR/Cas9 genome-editing technology. Mol. Ther. Nucl. Acids. 12, 267–274.
  29. Wang W., Ye C., Liu J., Zhang D., Kimata J.T., Zhou P. (2014) CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection. PLoS One. 9, e115987.
  30. Scott T., Urak R., Soemardy C., Morris K.V. (2019) Improved Cas9 activity by specific modifications of the tracrRNA. Sci. Rep. 9, 16104.
  31. Cho S.W., Kim S., Kim Y., Kweon J., Kim H.S., Bae S., Kim J.S. (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141.
  32. Gao Z., Herrera-Carrillo E., Berkhout B. (2018) Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA. RNA Biol. 15, 1458–1467.
  33. Liu Z., Liang J., Chen S., Wang K., Liu X., Liu B., Xia Y., Guo M., Zhang X., Sun G., Tian G. (2020) Genome editing of CCR5 by AsCas12a renders CD4+ T cells resistance to HIV-1 infection. Cell Biosci. 10, 85.
  34. Ratti V., Nanda S., Eszterhas S.K., Howell A.L., Wallace D.I. (2020) A mathematical model of HIV dynamics treated with a population of gene-edited haematopoietic progenitor cells exhibiting threshold phenomenon. Math. Med. Biol. 37, 212–242.
  35. Casini A., Olivieri M., Petris G., Montagna C., Reginato G., Maule G., Lorenzin F., Prandi D., Romanel A., Demichelis F., Inga A., Cereseto A. (2018) A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271.
  36. Lee J.K., Jeong E., Lee J., Jung M., Shin E., Kim Y.H., Lee K., Jung I., Kim D., Kim S., Kim J.S. (2018) Directed evolution of CRISPR/Cas9 to increase its specificity. Nat. Commun. 9, 3048.
  37. Tycko J., Myer V.E., Hsu P.D. (2016) Methods for optimizing CRISPR/Cas9 genome editing specificity. Mol. Cell. 63, 355–370.
  38. Kulcsár P.I., Tálas A., Tóth E., Nyeste A., Ligeti Z., Welker Z., Welker E. (2020) Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5’G-extended sgRNAs. Nat. Commun. 11, 1223.
  39. Kleinstiver B.P., Pattanayak V., Prew M.S., Tsai S.Q., Nguyen N.T., Zheng Z., Joung J.K. (2016) High-fidelity CRISPR/Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 529, 490–495.
  40. 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.
  41. Labun K., Montague T.G., Krause M., Torres Cleuren Y.N., Tjeldnes H., Valen E. (2019) CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucl. Acids Res. 47, W171–W174.
  42. 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, 475–485.
  43. GitHub/lioj/bioinformatics/offTargetPipeline at master. Доступен онлайн: https://github.com/lioj/bioinformatics/tree/master/offTargetPipeline (Проверено 07.05.2024).
  44. Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W., Donovan K.F., Smith I., Tothova Z., Wilen C., Orchard R., Virgin H.W., Listgarten J., Root D.E. (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR/Cas9. Nat. Biotechnol. 34, 184–191.
  45. Hsu P.D., Scott D.A., Weinstein J.A., Ran F.A., Konermann S., Agarwala V., Li Y., Fine E.J., Wu X., Shalem O., Cradick T.J., Marraffini L.A., Bao G., Zhang F. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832.
  46. Labuhn M., Adams F.F., Ng M., Knoess S., Schambach A., Charpentier E.M., Schwarzer A., Mateo J.L., Klusmann J.H., Heckl D. (2018) Refined sgRNA efficacy prediction improves large- and small-scale CRISPR/Cas9 applications. Nucl. Acids Res. 46, 1375–1385.
  47. Suzuki K., Tsunekawa Y., Hernandez-Benitez R., Wu J., Zhu J., Kim E.J., Hatanaka F., Yamamoto M., Araoka T., Li Z., Kurita M., Hishida T., Li M., Aizawa E., Guo S., Chen S., Goebl A., Soligalla R.D., Qu J., Jiang T., Fu X., Jafari M., Esteban C.R., Berggren W.T., Lajara J., Nuñez-Delicado E., Guillen P., Campistol J.M., Matsuzaki F., Liu G.H., Magistretti P., Zhang K., Callaway E.M., Zhang K., Belmonte J.C.I. (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature. 540, 144–149.
  48. Concordet J.P., Haeussler M. (2018) CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucl. Acids Res. 46(W1), W242–W245.

Supplementary files

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2. Fig. 1. gRNA efficiency. Average values ​​obtained in three independent experiments and standard deviations are shown. The dotted line shows the cutoff value used to select gRNAs whose off-target activity was determined in subsequent experiments.

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3. Fig. 2. Mutation frequencies in 63 predicted off-target regions of SpCas9 (a) and SpCas9-HF1-plus (b). Data were obtained from two independent experiments. *p-value < 0.001 by Fisher's exact test compared to control.

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5. Table S5
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