一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统

doi:10.16288/j.yczz.22-118

遗传 ›› 2022, Vol. 44 ›› Issue (8): 708-719.doi: 10.16288/j.yczz.22-118

一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统

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()

西北农林科技大学动物科技学院,杨凌 712100

收稿日期:2022-04-05 修回日期:2022-05-11 出版日期:2022-08-20 发布日期:2022-05-23
通讯作者: 徐坤,魏泽辉 E-mail:mshn15@163.com;weizehui7848@163.com;xukunas@nwafu.edu.cn
作者简介:张潇筠, 在读硕士研究生,专业方向：动物生物技术。E-mail: mshn15@163.com
基金资助:
国家自然科学基金项目编号(32172736);,陕西省重点研发计划项目编号(2021NY-027)

A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair

Xiaojun Zhang(), Kun Xu(), Juncen Shen, Lu Mu, Hongrun Qian, Jieyu Cui, Baoxia Ma, Zhilong Chen, Zhiying Zhang, Zehui Wei()

College of Animal Science and Technology, Northwest A&F University, Yangling 71200, China

Received:2022-04-05 Revised:2022-05-11 Online:2022-08-20 Published:2022-05-23
Contact: Xu Kun,Wei Zehui E-mail:mshn15@163.com;weizehui7848@163.com;xukunas@nwafu.edu.cn
Supported by:
the National Natural Science Foundation of China(32172736);the Shaanxi Key R&D Program(2021NY-027)

摘要/Abstract

摘要：

近年来,CRISPR基因编辑及衍生技术迅速发展,在生命科学、生物医学研究以及动植物育种领域得到了广泛应用。基于DNA双链断裂(double-stranded break, DSB)同源指导修复(homology-directed repair, HDR)机制的基因敲入和点编辑是基因编辑的重要策略,但效率偏低亟待提高。本文提出了驱动供体DNA富集至DSB处以提高HDR效率的新策略,并设计了一套CRISPR/Cas9-Gal4BD 供体适配基因编辑系统(donor adapting system, DAS)。该系统主要利用Gal4 DNA结合域(Gal4 binding domain, Gal4BD)作为配体蛋白与Cas9融合表达,将Gal4BD结合序列(Gal4 binding sequence, Gal4BS)作为受体序列与双链DNA (double-stranded DNA, dsDNA)供体结合,以期提高HDR效率。使用HEK293T-HDR.GFP报告细胞系的初步研究结果表明当dsDNA供体同源臂在一定长度(100~60 bp)时该系统能够提高HDR效率2~4倍。进一步的优化研究表明,融合端口和融合使用连接子(linker)的选择会影响Cas9表达效果及活性,而GGS5作为Cas9-Gal4BD融合的连接子则影响较小。同时,本研究还发现Gal4BS-dsDNA供体的差异化设计也会影响HDR效率,将Gal4BS添加到dsDNA供体5′-端的效果最佳。综上所述,本研究利用CRISPR/Cas9-Gal4BD DAS在AAVS1和EMX1位点上实现了HDR编辑效率的提高,为进一步利用该系统进行动物分子设计育种研究提供了参考和借鉴。

关键词: CRISPR/Cas9, 基因编辑, 供体DNA, 供体适配, 同源指导修复

Abstract:

The fast-rising CRISPR-derived gene editing technologies has been widely used in the fields of life science and biomedicine, as well as plant and animal breeding. However, the efficiency of homology-directed repair (HDR), an important strategy for gene knock-in and base editing, remains to be improved. In this study, we came up with the term Donor Adapting System (DAS) to summarize those CRISPR/Cas9 systems modified with adaptor for driving aptamer-fused donor DNA. A set of CRISPR/Cas9-Gal4BD DAS was designed in our study. In this system, Gal4 DNA binding domain (Gal4BD) is used as adaptor to fuse with Cas9 protein, and Gal4 binding sequence (Gal4BS) is used as aptamer to bind to the double-stranded DNA (dsDNA) donor, in order to improve the HDR efficiency. Preliminary results from the HEK293T-HDR.GFP reporter cell line show that the HDR editing efficiency could be improved up to 2-4 times when donor homologous arms under certain length (100-60 bp). Further optimization results showed that the choice of fusion port and fusion linker would affect the expression and activity of Cas9, while the Cas9-Gal4BD fusion with a GGS5 linker was the prior choice. In addition, the HDR efficiency was likely dependent on the aptamer-dsDNA donor design, and single Gal4BD binding sequence (BS) addition to the 5′-end of intent dsDNA template was suggested. Finally, we achieved enhanced HDR editing on the endogenous AAVS1 and EMX1 sites by using the CRISPR/Gal4BD-Cas9 DAS, which we believe can be applied to facilitate animal molecular design breeding in the future.

Key words: CRISPR/Cas9, gene editing, donor DNA, donor adapting, homology-directed repai

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[J]. 遗传, 2022, 44(8): 708-719.

Xiaojun Zhang, Kun Xu, Juncen Shen, Lu Mu, Hongrun Qian, Jieyu Cui, Baoxia Ma, Zhilong Chen, Zhiying Zhang, Zehui Wei. A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair[J]. Hereditas(Beijing), 2022, 44(8): 708-719.

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参考文献 51

[1]	Zhou SW, Yu HH, Zhao XE, Cai B, Ding Q, Huang Y, Li YX, Li Y, Niu YY, Lei AM, Kou QF, Huang XX, Petersen B, Ma BH, Chen YL, Wang XL. Generation of gene-edited sheep with a defined Booroola fecundity gene (FecB ^B) mutation in bone morphogenetic protein receptor type 1B (BMPR1B) via clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9. Reprod Fertil Dev, 2018, 30(12): 1616-1621. doi: 10.1071/RD18086
[2]	Niu YY, Zhao XE, Zhou JK, Li Y, Huang Y, Cai B, Liu YT, Ding Q, Zhou SW, Zhao J, Zhou GX, Ma BH, Huang XX, Wang XL, Chen YL.Efficient generation of goats with defined point mutation (I397V) in GDF9 through CRISPR/Cas9. Reprod Fertil Dev, 2018, 30(2): 307-312. doi: 10.1071/RD17068
[3]	Park KE, Kaucher AV, Powell A, Waqas MS, Sandmaier SES, Oatley MJ, Park CH, Tibary A, Donovan DM, Blomberg LA, Lillico SG, Whitelaw CBA, Mileham A, Telugu BP, Oatley JM. Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene. Sci Rep, 2017, 7: 40176. doi: 10.1038/srep40176
[4]	Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014, 157(6): 1262-1278. doi: 10.1016/j.cell.2014.05.010
[5]	Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol, 2015, 33(5): 543-548. doi: 10.1038/nbt.3198
[6]	Decottignies A. Alternative end-joining mechanisms: a historical perspective. Front Genet, 2013, 4: 48. doi: 10.3389/fgene.2013.00048 pmid: 23565119
[7]	Vasquez KM, Marburger K, Intody Z, Wilson JH. Manipulating the mammalian genome by homologous recombination. Proc Natl Acad Sci USA, 2001, 98(15): 8403-10. doi: 10.1073/pnas.111009698
[8]	Ruff P, Koh KD, Keskin H, Pai RB, Storici F. Aptamer-guided gene targeting in yeast and human cells. Nucleic Acids Res, 2014, 42(7): e61. doi: 10.1093/nar/gku101
[9]	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. doi: 10.7554/eLife.33761
[10]	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. doi: 10.1038/nbt.4166
[11]	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. doi: 10.1038/cr.2017.29
[12]	Carlson-Stevermer J, Abdeen AA, Kohlenberg L, Goedland M, Molugu K, Lou M, Saha K. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat Commun, 2017, 8(1): 1711. doi: 10.1038/s41467-017-01875-9 pmid: 29167458
[13]	Aird EJ, Lovendahl KN, Martin AS, Harris RS, Gordon WR. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun Biol, 2018, 1: 54. doi: 10.1038/s42003-018-0054-2
[14]	Ali Z, Shami A, Sedeek K, Kamel R, Alhabsi A, Tehseen M, Hassan N, Butt H, Kababji A, Hamdan SM, Mahfouz MM. Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice. Commun Biol, 2020, 3(1): 44. doi: 10.1038/s42003-020-0768-9
[15]	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. doi: 10.1016/j.jbc.2021.100525
[16]	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. doi: 10.1093/nar/gkaa779
[17]	Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol, 2015, 33(5): 538-542. doi: 10.1038/nbt.3190 pmid: 25798939
[18]	Hu Z, Shi Z, Guo X, Jiang B, Wang G, Luo D, Chen Y, Zhu YS. Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR-Cas9 and ssODN in human cancer cells. Cell Biosci. 2018 19(8):12.
[19]	Riesenberg S, Maricic T. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. Nat Commun, 2018, 9(1): 2164. doi: 10.1038/s41467-018-04609-7 pmid: 29867139
[20]	Riesenberg S, Chintalapati M, Macak D, Kanis P, Maricic T, Pääbo S. Simultaneous precise editing of multiple genes in human cells. Nucleic Acids Res. 2019 47(19):e116. doi: 10.1093/nar/gkz669
[21]	Robert F, Barbeau M, Éthier S, Dostie J, Pelletier J. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med, 2015, 7(1): 93. doi: 10.1186/s13073-015-0215-6
[22]	Li G, Liu D, Zhang X, Quan R, Zhong C, Mo J, Huang Y, Wang H, Ruan X, Xu Z, Zheng E, Gu T, Hong L, Li Z, Wu Z, Yang H. Suppressing Ku70/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts. Int J Biochem Cell Biol, 2018, 99: 154-160. doi: 10.1016/j.biocel.2018.04.011
[23]	Nambiar TS, Billon P, Diedenhofen G, Hayward SB, Taglialatela A, Cai KH, Huang JW, Leuzzi G, Cuella-Martin R, Palacios A, Gupta A, Egli D, Ciccia A. Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant. Nat Commun, 2019, 10(1): 3395. doi: 10.1038/s41467-019-11105-z pmid: 31363085
[24]	Paulsen BS, Mandal PK, Frock RL, Boyraz B, Yadav R, Upadhyayula S, Gutierrez-Martinez P, Ebina W, Fasth A, Kirchhausen T, Talkowski ME, Agarwal S, Alt FW, Rossi DJ. Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing. Nat Biomed Eng, 2017, 1(11): 878-888. doi: 10.1038/s41551-017-0145-2 pmid: 31015609
[25]	Tran NT, Bashir S, Li X, Rossius J, Chu VT, Rajewsky K, Kühn R. Enhancement of precise gene editing by the association of Cas9 with homologous recombination Factors. Front Genet, 2019, 10: 365. doi: 10.3389/fgene.2019.00365
[26]	Hackley CR. A novel set of Cas9 fusion proteins to stimulate homologous recombination: Cas9-HRs. CRISPR J, 2021, 4(2): 253-263. doi: 10.1089/crispr.2020.0034 pmid: 33876961
[27]	Shao SM, Ren CH, Liu ZT, Bai YC, Chen ZL, Wei ZH, Wang X, Zhang ZY, Xu K.Enhancing CRISPR/Cas9- mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52. Int J Biochem Cell Biol, 2017, 92: 43-52. doi: 10.1016/j.biocel.2017.09.012
[28]	Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol, 2016, 34(3): 339-344. doi: 10.1038/nbt.3481
[29]	Shy BR, Vykunta V, Ha A, Roth TL, Talbot A, Nguyen DN, Chen YY, Blaeschke F, Vedova S, Mamedov MR, Chung JY, Li H, Wolf J, Martin TG, Ye LM, Eyquem J, Esensten JH, Marson A. Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing. bioRxiv, 2021, doi: 10.1101/2021.09.02.458799. doi: 10.1101/2021.09.02.458799
[30]	Cruz-Becerra G, Kadonaga JT. Enhancement of homology-directed repair with chromatin donor templates in cells. eLife, 2020, 9: e55780. doi: 10.7554/eLife.55780
[31]	Hirotsune S, Kiyonari H, Jin MY, Kumamoto K, Yoshida K, Shinohara M, Watanabe H, Wynshaw-Boris A, Matsuzaki F. Enhanced homologous recombination by the modulation of targeting vector ends. Sci Rep, 2020, 10(1): 2518. doi: 10.1038/s41598-020-58893-9 pmid: 32054870
[32]	Liang X, Potter J, Kumar S, Ravinder N, Chesnut JD. Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. J Biotechnol, 2017, 241: 136-146. doi: 10.1016/j.jbiotec.2016.11.011
[33]	Nguyen DN, Roth TL, Li PJ, Chen PA, Apathy R, Mamedov MR, Vo LT, Tobin VR, Goodman D, Shifrut E, Bluestone JA, Puck JM, Szoka FC, Marson A. Polymer- stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat Biotechnol, 2020, 38(1): 44-49. doi: 10.1038/s41587-019-0325-6 pmid: 31819258
[34]	Ling X, Xie B, Gao X, Chang L, Zheng W, Chen H, Huang Y, Tan L, Li M, Liu T. Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates. Sci Adv, 2020, 6(15): eaaz0051. doi: 10.1126/sciadv.aaz0051
[35]	Lomova A, Clark DN, Campo-Fernandez B, Flores- Bjurström C, Kaufman ML, Fitz-Gibbon S, Wang XY, Miyahira EY, Brown D, DeWitt MA, Corn JE, Hollis RP, Romero Z, Kohn DB. Improving gene editing outcomes in human hematopoietic stem and progenitor cells by temporal control of DNA repair. Stem Cells, 2019, 37(2): 284-294.
[36]	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. doi: 10.1038/s41467-019-13787-x
[37]	Zhang JP, Li XL, Li GH, Chen W, Arakaki C, Botimer GD, Baylink D, Zhang L, Wen W, Fu YW, Xu J, Chun N, Yuan W, Cheng T, Zhang XB. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol, 2017, 18(1): 35. doi: 10.1186/s13059-017-1164-8
[38]	Gutschner T, Haemmerle M, Genovese G, Draetta GF, Chin L. Post-translational regulation of Cas9 during G1 enhances homology-directed repair. Cell Rep, 2016, 14(6): 1555-1566. doi: S2211-1247(16)00040-1 pmid: 26854237
[39]	Matsumoto D, Tamamura H, Nomura W. A cell cycle- dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy. Commun Biol, 2020, 3(1): 601-601. doi: 10.1038/s42003-020-01340-2 pmid: 33097793
[40]	Li G, Zhang X, Zhong C, Mo J, Quan R, Yang J, Liu D, Li Z, Yang H, Wu Z. Small molecules enhance CRISPR/ Cas9-mediated homology-directed genome editing in primary cells. Sci Rep, 2017, 7(1): 8943. doi: 10.1038/s41598-017-09306-x
[41]	Takayama K, Igai K, Hagihara Y, Hashimoto R, Hanawa M, Sakuma T, Tachibana M, Sakurai F, Yamamoto T, Mizuguchi H. Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system. Nucleic Acids Res, 2017, 45(9): 5198-5207. doi: 10.1093/nar/gkx130 pmid: 28334759
[42]	Li GL, Zhang XW, Wang HQ, Liu DW, Li ZC, Wu ZF, Yang HQ. Increasing CRISPR/Cas9-mediated homology- directed DNA repair by histone deacetylase inhibitors. Int J Biochem Cell Biol, 2020, 125: 105790. doi: 10.1016/j.biocel.2020.105790
[43]	Keegan L, Gill G, Ptashne M. Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science, 1986, 231(4739): 699-704. pmid: 3080805
[44]	Marmorstein R, Carey M, Ptashne M, Harrison SC. DNA recognition by GAL4: structure of a protein-DNA complex. Nature, 1992, 356(6368): 408-414. doi: 10.1038/356408a0
[45]	Lohr D, Venkov P, Zlatanova J. Transcriptional regulation in the yeast GAL gene family: a complex genetic network. FASEB J, 1995, 9(9): 777-787. pmid: 7601342
[46]	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 thermophilus. Cell Mol Life Sci, 2015, 72(2): 383-399. doi: 10.1007/s00018-014-1679-z
[47]	Liang SD, Marmorstein R, Harrison SC, Ptashne M. DNA sequence preferences of GAL4 and PPR1: how a subset of Zn2 Cys6 binuclear cluster proteins recognizes DNA. Mol Cell Biol, 1996, 16(7): 3773-3780. doi: 10.1128/MCB.16.7.3773 pmid: 8668194
[48]	Bram RJ, Lue NF, Kornberg RD. A GAL family of upstream activating sequences in yeast: roles in both induction and repression of transcription. EMBO J, 1986, 5(3): 603-608. doi: 10.1002/j.1460-2075.1986.tb04253.x pmid: 3011415
[49]	Selleck SB, Majors JE. In vivo DNA-binding properties of a yeast transcription activator protein. Mol Cell Biol, 1987, 7(9): 3260-3267. doi: 10.1128/mcb.7.9.3260-3267.1987 pmid: 3313011
[50]	Yan NN, Sun YS, Fang YY, Deng JR, Mu L, Xu K, Mymryk JS, Zhang ZY. A universal surrogate reporter for efficient enrichment of CRISPR/Cas9-mediated homology- directed repair in mammalian cells. Mol Ther Nucleic Acids, 2020, 19: 775-789. doi: 10.1016/j.omtn.2019.12.021
[51]	Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M, Aizawa E, Guo SC, Chen S, Goebl A, Soligalla RD, Qu J, Jiang TS, Fu X, Jafari M, Esteban CR, Berggren WT, Lajara J, Nuñez-Delicado E, Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang K, Callaway EM, Zhang K, Belmonte JCI. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature, 2016, 540(7631): 144-149. doi: 10.1038/nature20565

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分类	具体策略	检测所用细胞类型	不同类型供体提升效果		代表性参考文献
分类	具体策略	检测所用细胞类型	dsDNA	ssDNA	代表性参考文献
抑制NHEJ 通路	抑制DNA连接酶4	HEK293/K562/MEF/DC2.4/MelJuSo/HCT-116	2.4~19倍	~3倍	[5,17,18]
	抑制蛋白激酶催化亚基	HEK293/iPSC	2~5倍	1.6~3.3倍	[5,19,20]
	抑制Ku蛋白	HEK293/PFF	2~3倍	~2.4倍	[21,22]
	抑制53BP1	HEK293/Hela/K562/U2OS/LCL B/iPSC	1.4~3倍	1.3~3.3倍	[23,24]
增强HDR 通路	共表达CtIP转录因子	HEK293/iPSC	1.5~14.9倍	—	[25]
	共表达外切核酸酶1	K562/A549/H1299	2~2.5倍	—	[26]
	共表达Rad51、Rad52	HEK293/PK15/iPSC	2~6倍	1.4~2.4倍	[24,27]
优化供体形式	ssDNA供体优化	HEK293/U2OS/T cells	—	~3倍	[28,29]
	环状dsDNA供体优化	HEK293/HeLa/MCF10A/Embryo	1.1~18倍	—	[30,31]
	线性dsDNA供体优化	HEK293/iPSC	2~10倍	1.4~10倍	[32,33]
	供体与DBS共定位	HEK293	3~6倍	1.6~18倍	[11,12,34]
控制打靶时效	调节细胞周期	HEK293/iPSC/T cells	1.7~6倍	—	[35-37]
控制打靶时效	Cas9定时生效	HEK293/Two-cell embryo	1.87~10倍	1~5倍	[38,39]
其他机制	小分子化合物	HEK293/PFF/iPSC	2~3倍	2~10倍	[40]
其他机制	染色质状态	PFF/hES/iPSC/Embryo	~2倍	2-3倍	[41,42]

系统简称	融合端	Linker	适配配体	配体大小	适配受体	受体大小	供体形式	参考文献
Cas9-SNAP/ ssDNA	Cas9 C-terminus	Undeclared	SNAP-tag	181 aa	O6-BG	~241 (MW)	ssDNA	[9]
Cas9-Avidin/ dsDNA	Cas9	SGSETPGTSESATPES (16 aa)	Monomeric Streptavidin	114 aa	Biotin	~244 (MW)	dsDNA	[10]
Cas9-Avidin/ ssDNA	Cas9 C-terminus	SGSETPGTSESATPES (16 aa)	Avidin	152 aa	Biotin	~244 (MW)	ssDNA	[11]
sgRNA-S1m/ ssDNA	sgRNA	S1m*	Recombinant Streptavidin	159 aa	Biotin	~244 (MW)	ssDNA	[12]
Cas9-PVC/ ssDNA	Cas9 C-terminus	H4-2	Porcine Circovirus 2 (PCV) Rep	109 aa	PCV.BS	13 nt	ssDNA	[13]
Cas9-VirD2/ ssDNA (Plant editing)	Cas9	Undeclared	VirD2	455 aa	T-DNA.BS	25 nt	ssDNA	[14]
Cas9-THAP11/ dsDNA	Cas9 C-terminus	SGSETPGTSESATPES (16 aa)	THAP11	105 aa	THAP11.BS	19 bp	dsDNA	[15]
Cas9-N57/ dsDNA	Cas9 C-terminus	GGGGGSGGGGSGGGGSGGGGSLDPGGGGSG (30 aa)	N57	57 aa	N57.BS	292 bp	dsDNA	[16]
Cas9-Gal4BD/ dsDNA	Cas9 C-terminus	5×GGS (15 aa)	Gal4BD	146 aa	Gal4BD.BS	17 bp	dsDNA	This study

一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统

A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair

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[1]	卞中, 曹东平, 庄文姝, 张舒玮, 刘巧泉, 张林. 水稻分子设计育种启示：传统与现代相结合[J]. 遗传, 2023, 45(9): 718-740.
[2]	王秉政, 张超, 张佳丽, 孙锦. 利用单转录本表达Cas9和sgRNA条件性编辑果蝇基因组[J]. 遗传, 2023, 45(7): 593-601.
[3]	吴仲胜, 高誉, 杜勇涛, 党颂, 何康敏. CRISPR-Cas9基因编辑技术对细胞内源蛋白进行荧光标记的实验操作[J]. 遗传, 2023, 45(2): 165-175.
[4]	刘梅珍, 王立人, 李咏梅, 马雪云, 韩红辉, 李大力. 利用CRISPR/Cas9技术构建基因编辑大鼠模型[J]. 遗传, 2023, 45(1): 78-87.
[5]	张充, 魏子璇, 王敏, 陈瑶生, 何祖勇. 利用CRISPR/Cas9在人类黑色素瘤细胞中编辑MC1R与功能分析[J]. 遗传, 2022, 44(7): 581-590.
[6]	刘尧, 周先辉, 黄舒泓, 王小龙. 引导编辑：突破碱基编辑类型的新技术[J]. 遗传, 2022, 44(11): 993-1008.
[7]	韩玉婷, 许博文, 李羽童, 卢心怡, 董习之, 邱雨浩, 车沁耘, 朱芮葆, 郑丽, 李孝宸, 司绪, 倪建泉. 模式动物果蝇的基因调控前沿技术[J]. 遗传, 2022, 44(1): 3-14.
[8]	王海涛, 李亭亭, 黄勋, 马润林, 刘秋月. 遗传修饰技术在绵羊分子设计育种中的应用[J]. 遗传, 2021, 43(6): 580-600.
[9]	杨光武, 田嫄. 果蝇F-box基因Ppa促进脂肪储存[J]. 遗传, 2021, 43(6): 615-622.
[10]	彭定威, 李瑞强, 曾武, 王敏, 石翾, 曾检华, 刘小红, 陈瑶生, 何祖勇. 编辑MSTN半胱氨酸节基元促进两广小花猪肌肉生长[J]. 遗传, 2021, 43(3): 261-270.
[11]	王娜, 甲芝莲, 吴强. RFX5调控原钙粘蛋白α基因簇的表达[J]. 遗传, 2020, 42(8): 760-774.
[12]	李国玲, 杨善欣, 吴珍芳, 张献伟. 提高CRISPR/Cas9介导的动物基因组精确插入效率研究进展[J]. 遗传, 2020, 42(7): 641-656.
[13]	陈赢男, 陆静. CRISPR/Cas9系统在林木基因编辑中的应用[J]. 遗传, 2020, 42(7): 657-668.
[14]	李霞, 施皖, 耿立召, 许建平. CRISPR/Cas核糖核蛋白介导的植物基因组编辑[J]. 遗传, 2020, 42(6): 556-564.
[15]	秦瑞英, 魏鹏程. Prime editing引导植物基因组精确编辑新局面[J]. 遗传, 2020, 42(6): 519-523.