Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing
Received date: 2024-01-07
Revised date: 2024-03-22
Online published: 2024-03-27
Supported by
Scientific and Technological Innovation 2030?Major Project of Agricultural Biological Breeding(2023ZD04074);Scientific and Technological Innovation 2030?Major Project of Agricultural Biological Breeding(2023ZD04051)
Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3′-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.
Key words： gene editing; gene knock-in; CRISPR/Cas9; donor adapting; homology-directed repair
Ma Baoxia, Yang Sen, Lyu Ming, Wang Yuren, Chang Liye, Han Yifan, Wang Jiangang, Guo Yang, Xu Kun . Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing[J]. Hereditas(Beijing), 2024 , 46(6) : 466 -477 . DOI: 10.16288/j.yczz.23-273
| [1] | Zhang HX, Zhang Y, Yin H. Genome editing with mRNA encoding ZFN, TALEN, and Cas9. Mol Ther, 2019, 27(4): 735-746. |
| [2] | Carroll D. Genome engineering with targetable nucleases. Annu Rev Biochem, 2014, 83: 409-439. |
| [3] | Shalem O, Sanjana NE, Zhang F. High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet, 2015, 16(5): 299-311. |
| [4] | Wang JY, Doudna JA. CRISPR technology: a decade of genome editing is only the beginning. Science, 2023, 379(6629): eadd8643. |
| [5] | Fu YW, Dai XY, Wang WT, Yang ZX, Zhao JJ, Zhang JP, Wen W, Zhang F, Oberg KC, Zhang L, Cheng T, Zhang XB. Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res, 2021, 49(2): 969-985. |
| [6] | Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet, 2018, 19(12): 770-788. |
| [7] | Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol, 2020, 38(7): 824-844. |
| [8] | Mikkelsen NS, Bak RO. Enrichment strategies to enhance genome editing. J Biomed Sci, 2023, 30(1): 51. |
| [9] | Yang H, Ren SL, Yu SY, Pan HF, Li TD, Ge SX, Zhang J, Xia NS. Methods favoring homology-directed repair choice in response to CRISPR/Cas9 induced-double strand breaks, Int J Mol Sci, 2020, 21(18): 6461. |
| [10] | Sun WL, Liu H, Yin WH, Qiao J, Zhao XK, Liu Y. Strategies for enhancing the homology-directed repair efficiency of CRISPR-Cas systems. CRISPR J, 2022, 5(1): 7-18. |
| [11] | Shy BR, Vykunta VS, Ha A, Talbot A, Roth TL, Nguyen DN, Pfeifer WG, Chen YY, Blaeschke F, Shifrut E, Vedova S, Mamedov MR, Chung JYJ, Li H, Yu R, Wu D, Wolf J, Martin TG, Castro CE, Ye L, Esensten JH, Eyquem J, Marson A. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat Biotechnol, 2023, 41(4): 521-531. |
| [12] | Riesenberg S, Kanis P, Macak D, Wollny D, Düsterh?ft D, Kowalewski J, Helmbrecht N, Maricic T, P??bo S. Efficient high-precision homology-directed repair-dependent genome editing by HDRobust. Nat Methods, 2023, 20(9): 1388-1399. |
| [13] | Ma M, Zhuang FF, Hu XB, Wang BL, Wen XZ, Ji JF, Xi JZJ. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res, 2017, 27(4): 578-581. |
| [14] | Savic N, Ringnalda FC, Lindsay H, Berk C, Bargsten K, Li YZ, Neri D, Robinson MD, Ciaudo C, Hall J, Jinek M, Schwank G. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology- directed repair. eLife, 2018, 7: e33761. |
| [15] | Gu B, Posfai E, Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol, 2018, 36(7): 632-637. |
| [16] | Aird EJ, Lovendahl KN, St Martin A, Harris RS, Gordon WR. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun Biol, 2018, 1: 54. |
| [17] | Lee K, Mackley VA, Rao A, Chong AT, Dewitt MA, Corn JE, Murthy N. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. eLife, 2017, 6: e25312. |
| [18] | Li GL, Wang HQ, Zhang XW, Wu ZF, Yang HQ. A Cas9-transcription factor fusion protein enhances homology-directed repair efficiency. J Biol Chem, 2021, 296: 100525. |
| [19] | Ma SF, Wang XL, Hu YF, Lv J, Liu CF, Liao KT, Guo XH, Wang D, Lin Y, Rong ZL. Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain. Nucleic Acids Res, 2020, 48(18): 10590-10601. |
| [20] | Wienert B, Nguyen DN, Guenther A, Feng SJ, Locke MN, Wyman SK, Shin J, Kazane KR, Gregory GL, Carter MAM, Wright F, Conklin BR, Marson A, Richardson CD, Corn JE. Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair. Nat Commun, 2020, 11(1): 2109. |
| [21] | Zhang XJ, Xu K, Shen JC, Mu L, Qian HR, Cui JY, Ma BX, Chen ZL, Zhang ZY, Wei ZH. A CRISPR/Cas9- Gal4BD donor adapting system for enhancing homology- directed repair. Hereditas (Beijing), 2022, 44(8): 708-719. |
| 张潇筠, 徐坤, 沈俊岑, 穆璐, 钱泓润, 崔婕妤, 马宝霞, 陈知龙, 张智英, 魏泽辉. 一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统. 遗传, 2022, 44(8): 708-719. | |
| [22] | Ma BX, Cui JY, Qian HR, Zhang XJ, Yang S, Zhang QJ, Han YF, Zhang ZY, Wang JG, Xu K. A novel CRISPR/ Cas9-hLacI donor adapting system for dsDNA-templated gene editing. Chin J Biotechnol, 2023, 39(10): 4204-4218. |
| 马宝霞, 崔婕妤, 钱泓润, 张潇筠, 杨森, 张骐镜, 韩艺帆, 张智英, 王建刚, 徐坤. 一种新型的CRISPR/Cas9- hLacI双链DNA供体适配基因编辑系统. 生物工程学报, 2023, 39(10): 4204-4218. | |
| [23] | Kong XZ, Yin RH, Ning HM, Zheng WW, Dong XM, Yang Y, Xu FF, Li JJ, Zhan YQ, Yu M, Ge CH, Zhang JH, Chen H, Li CY, Yang XM. Effects of THAP11 on erythroid differentiation and megakaryocytic differentiation of K562 cells. PloS One, 2014, 9(3): e91557. |
| [24] | Hew BE, Sato R, Mauro D, Stoytchev I, Owens JB. RNA-guided piggyBac transposition in human cells. Synth Biol (Oxf), 2019, 4(1): ysz018. |
| [25] | Xu K, Ren CH, Liu ZT, Zhang T, Zhang TT, Li D, Wang L, Yan Q, Guo LJ, Shen JC, Zhang ZY. Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophiles. Cell Mol Life Sci, 2015, 72(2): 383-399. |
| [26] | Koch B, Nijmeijer B, Kueblbeck M, Cai Y, Walther N, Ellenberg J. Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing. Nat Protoc, 2018, 13(6): 1465-1487. |
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号
