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| HDACi and RS-1 Enhance CRISPR/Cas12i-mediated HDR Editing Efficiency |
| CHEN Qiu-Chong, LI Shang-Pu, MIAO Er-Yu, ZHOU Bing-Qian, WANG Xu, MENG Xiang-Yu, WANG Xiao-Long*, XU Kun* |
| College of Animal Science and Technology/International Joint Research Center of Animal Biobreeding, Ministry of Agriculture and Rural Affairs/Shaanxi Key Laboratory of Animal Genetic Breeding and Reproduction, Northwest A&F University, Yangling 712100, China |
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Abstract CRISPR/Cas12i is a recently developed CRISPR gene editing system by Chinese scholars, which has independent intellectual property rights and has been proven to have targeting efficiency comparable to the CRISPR/Cas9 system. Homology-directed repair (HDR) is one of the main repair mechanisms for double-stranded DNA breaks (DSBs). Gene editing based on the HDR mechanism can be used to correct any form of mutation in the genome, but it is often limited by the generally lower HDR efficiency in mammalian cells. In this study, the activity of the CRISPR/Cas12i system at different target sites in Homo sapiens embryonic kidney cell line HEK293T, the optimal dosage of single-stranded oligonucleotides (ssODN) donor templates for mediating HDR editing, and the appropriate concentration of small molecule drugs were verified through single-strand annealing (SSA) reporter experiment, dose gradient and concentration gradient experiment. The effects of adding different small molecule drugs on the HDR editing efficiency mediated by the CRISPR/Cas12i system in HEK293T cells and sheep (Ovis aries) fetal fibroblasts were then evaluated using flow cytometry sorting, genomic PCR, Sanger sequencing, and online prediction tools. The results showed that the CRISPR/Cas12i system exhibited high activity at 18 different target sites in HEK293T cells, with efficiencies around 80%, except 2 slightly lower sites. Different dosages and lengths of ssODN had some influence on HDR efficiency, and the appropriate concentrations of small molecule drugs varied slightly in different species and cell types. The addition of histone deacetylase inhibitor (HDACi) and RS-1 (C20H16Br2N2O3S) significantly enhanced the HDR editing efficiency mediated by the CRISPR/Cas12i system in both HEK293T cells and sheep fetal fibroblasts. Among them, RS-1 showed the least cytotoxicity and did not significantly reduce the insertion/deletion mutation (InDel) efficiency while improving HDR efficiency. Furthermore, Entinostat increased HDR editing efficiency by approximately 148 fold at the bone morphogenetic protein receptor 1B (BMPR1B) site in sheep fetal fibroblasts. In conclusion, the CRISPR/Cas12i system exhibited high activity and could mediate efficient and precise HDR editing using ssODN as a donor in model cells and primary sheep cells. The appropriate concentrations of HDACi and RS-1 could effectively improve HDR editing efficiency. This study provides reference and guidance for the application and popularization of the CRISPR/Cas12i gene editing system.
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Received: 11 December 2023
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Corresponding Authors:
* xiaolongwang@nwafu.edu.cn;xukunas@nwafu.edu.cn
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[1] 韩芙蓉, 王令, 徐坤, 等. 2015. 基于SSA修复机制和特异性核酸酶活性检测的双荧光报告载体系统的开发及应用[J]. 遗传, 37(10): 1053-1060.
(Han F R, Wang L, Xu K, et al.2015. Development and application of dual fluorescence reporter vector system based on SSA repair mechanism and specific nuclease activity detection[J]. Hereditas, 37(10): 1053-1060.)
[2] 杨初蕾, 张译元, 唐红, 等. 2023. FecB基因在绵羊繁殖性能上的研究进展[J]. 畜牧兽医学报, 54(04): 1370-1380.
(Yang C L, Zhang Z Y, Tang H, et al.2023. Research progress of FecB gene on reproductive performance of sheep[J]. Acta Veterinaria et Zootechnica Sinica, 54(04): 1370-1380.)
[3] Allen F, Crepaldi L, Alsinet C, et al.2018. Predicting the mutations generated by repair of Cas9-induced double-strand breaks[J]. Nature Biotechnology, 37: 64-72.
[4] Bai W, Huang M, Li C, et al.2023. The biological principles and advanced applications of DSB repair in CRISPR-mediated yeast genome editing[J]. Synthetic and Systems Biotechnology, 8(4): 584-596.
[5] Bhargava R, Onyango D O, Stark J M.2016. Regulation of single-strand annealing and its role in genome maintenance[J]. Trends in Genetics, 32(9): 566-575.
[6] Canny M D, Moatti N, Wan L C K, et al.2018. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency[J]. Nature Biotechnology, 36(1): 95-102.
[7] Chari R, Mali P, Moosburner M, et al.2015. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach[J]. Nature Methods, 12(9): 823-826.
[8] Chen Y, Hu Y, Wang X, et al.2022. Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing[J]. Innovation, 3(4): 100264.
[9] Choi H R, Lim H, Lee J H, et al.2021. Interruption of helicobacter pylori-induced NLRP3 inflammasome activation by chalcone derivatives[J]. Biomolecules & Therapeutics, 29(4): 410-418.
[10] Francis R, Mathilde B, Sylvain É, et al.2015. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing[J]. Genome Medicine, 7(1): 93.
[11] Gillmore J D, Gane E, Taubel J, et al.2021. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis[J]. New England Journal of Medicine, 385(6): 493-502.
[12] Haberland M, Montgomery R L, Olson E N.2009. The many roles of histone deacetylases in development and physiology: Implications for disease and therapy[J]. Nature Reviews. Genetics, 10(1): 32-42.
[13] Hendrickson E A.1997. Cell-cycle regulation of mammalian DNA double-strand-break repair[J]. American Journal of Human Genetics, 61(4): 795-800.
[14] Hu Z, Shi Z, Guo X, et al.2018. Ligase Ⅳ inhibitor SCR7 enhances gene editing directed by CRISPR-Cas9 and ssODN in human cancer cells[J]. Cell & Bioscience, 8: 12.
[15] Jang D E, Lee J Y, Lee J H, et al.2018. Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency[J]. Experimental & Molecular Medicine, 50(4): 1-9.
[16] Jayanthan A, Hofmann B, Meier-Stephenson V, et al.2019. Targeted Polo-like kinase inhibition combined with aurora kinase inhibition in pediatric acute leukemia cells[J]. Journal of Pediatric Hematology/Oncology, 41(6): e359-e370.
[17] Jayathilaka K, Sheridan S D, Bold T D, et al.2008. A chemical compound that stimulates the human homologous recombination protein RAD51[J]. Proceedings of the National Academy of Sciences of the USA, 105(41): 15848-15853.
[18] Komor A C, Badran A H, Liu D R.2017. CRISPR-based technologies for the manipulation of eukaryotic genomes[J]. Cell, 169(3): 559.
[19] Kouzarides T.2001. Chromatin modifications and their function[J]. Cell, 128(4): 693-705.
[20] Liu B, Chen S, Rose A, et al.2020. Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing[J]. Nucleic Acids Research, 48(2): 517-532.
[21] Liu J J, Orlova N, Oakes B L, et al.2019. CasX enzymes comprise a distinct family of RNA-guided genome editors[J]. Nature, 566(7743): 218-223.
[22] Makarova K S, Wolf Y I, Iranzo J, et al.2020. Evolutionary classification of CRISPR-Cas systems: A burst of class 2 and derived variants[J]. Nature Reviews Microbiology, 18(2): 67-83.
[23] Maruyama T, Dougan S K, Truttmann M C, et al.2015. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining[J]. Nature Biotechnology, 33(5): 538-542.
[24] McGaw C, Garrity A J, Munoz G Z, et al.2022. Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing[J]. Nature Communications, 13(1): 2833.
[25] Miura H, Gurumurthy C B, Sato T, et al.2015. CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA[J]. Scientific Reports, 5: 12799.
[26] Pinder J, Salsman J, Dellaire G.2015. Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing[J]. Nucleic Acids Research, 43(19): 9379-9392.
[27] Rahmani B, Kheirandish M H, Ghanbari S, et al.2023. Targeting DNA repair pathways with B02 and Nocodazole small molecules to improve CRIS-PITCh mediated cassette integration in CHO-K1 cells[J]. Scientific Reports, 13(1): 3116.
[28] Riesenberg S, Maricic T.2018. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells[J]. Nature Communications, 9(1): 2164.
[29] Seto E, Yoshida M.2014. Erasers of histone acetylation: The histone deacetylase enzymes[J]. Cold Spring Harbor Perspectives in Biology, 6(4): a018713.
[30] Shao S, Ren C, Liu Z, et al.2017. Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52[J]. International Journal of Biochemistry & Cell Biology, 92: 43-52.
[31] Shen M W, Arbab M, Hsu J Y, et al.2018. Predictable and precise template-free CRISPR editing of pathogenic variants[J]. Nature, 563(7733): 646-651.
[32] Teng F, Cui T, Feng G, et al.2018. Repurposing CRISPR-Cas12b for mammalian genome engineering[J]. Cell Discovery, 4: 63.
[33] Verma P, Greenberg R A.2016. Noncanonical views of homology-directed DNA repair[J]. Genes & Development, 30(10): 1138-1154.
[34] Yang D, Scavuzzo M A, Chmielowiec J, et al.2016. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases[J]. Scientific Reports, 6: 21264.
[35] Yeh C D, Richardson C D, Corn J E.2019. Advances in genome editing through control of DNA repair pathways[J]. Nature Cell Biology, 21(12): 1468-1478.
[36] Yoshimi K, Kunihiro Y, Kaneko T, et al.2016. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes[J]. Nature Communications, 7: 10431.
[37] Yu C, Liu Y, Ma T, et al.2015. Small molecules enhance CRISPR genome editing in pluripotent stem cells[J]. Cell Stem Cell, 16(2): 142-147.
[38] Yu W, Lescale C, Babin L, et al.2020. Repair of G1 induced DNA double-strand breaks in S-G2/M by alternative NHE[J][J]. Nature Communications, 11(1): 5239.
[39] Zetsche B, Gootenberg J S, Abudayyeh O O, et al.2015. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system[J]. Cell, 163(3): 759-771.
[40] Zhang G, Liu Y, Huang S, et al.2022. Enhancement of prime editing via xrRNA motif-joined pegRNA[J]. Nature Communications, 13(1): 1856.
[41] Zhang H, Kong X, Xue M, et al.2023. An engineered xCas12i with high activity, high specificity, and broad PAM range[J]. Protein & Cell, 14(7): 538-543.
[42] Zhang J P, Yang Z X, Zhang F, et al.2021. HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs[J]. Science China. Life Sciences, 4(9): 1449-1462. |
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