双定位信号增强工程化蛋白线粒体靶向性呈递

doi:10.16288/j.yczz.24-171

[an error occurred while processing this directive]

Hereditas(Beijing) ›› 2024, Vol. 46 ›› Issue (11): 937-946.doi: 10.16288/j.yczz.24-171

• Research Article • Previous Articles Next Articles

Dual-localization signals enhance mitochondrial targeted presentation of engineered proteins

Bingqian Zhou¹(), Shangpu Li¹, Xu Wang¹, Xiangyu Meng¹, Jingrong Deng¹, Jinliang Xing²(), Jiangang Wang¹, Kun Xu¹()

1. College of Animal Science and Technology, Northwest A&F University, Yangling 71200, China
2. State Key Laboratory of Tumour Biology, Fourth Military Medical University, Xi'an 710000, China

Received:2024-06-12 Revised:2024-09-01 Online:2024-11-20 Published:2024-09-04
Contact: Jinliang Xing, Kun Xu E-mail:2770998747@qq.com;xingjl@fmmu.edu.cn;xukun@nwafu.edu.cn
Supported by:
Biological Breeding-Major Projects(2023ZD04074);Biological Breeding-Major Projects(2023ZD04051);Open Fund Project from State Key Laboratory of Tumor Biology-China(CBSKL2022ZDKF11)

Abstract

Abstract:

Effective delivery of engineered proteins into mitochondria is of great significance for developing efficient mitochondrial DNA editing tools and realizing accurate treatment of mitochondrial diseases. Here, the candidate genes, eGFP and Cas9, were engineered with different mitochondrial localization signal (MLS) sequences introduced at their up- or/and down-streams. The corresponding expression vectors for the engineered proteins were constructed respectively, and HEK293T cells were transfected with these vectors. The fluorescence colocalization and Western blotting assays were used to analyze the mitochondrial targeting presentation effect of different engineered proteins. The results demonstrated that the daul-MLS modification of the eGFP and Cas9 proteins significantly improved the efficiency of mitochondrial targeted presentation, compared with the engineered proteins with single MLS added. Hence, it is speculated that dual MLS strategy can enhance the mitochondrial targeting of engineered proteins, which lays a theoretical foundation for the future development of efficient mitochondrial DNA editing tools.

Key words: mitochondrial diseases, mtDNA editing, protein engineering, mitochondrial localization signal, mitochondrial targeting

Bingqian Zhou, Shangpu Li, Xu Wang, Xiangyu Meng, Jingrong Deng, Jinliang Xing, Jiangang Wang, Kun Xu. Dual-localization signals enhance mitochondrial targeted presentation of engineered proteins[J]. Hereditas(Beijing), 2024, 46(11): 937-946.

Figures/Tables 6

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Table 1

References 28

[1]	Mcbride HM, Neuspiel M, Wasiak S. Mitochondria: more than just a powerhouse. Curr Biol, 2006, 16(14): R551- R560.
[2]	Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJ, Staden R, Young IG. Sequence and organization of the human mitochondrial genome. Nature, 1981, 290(5806): 457-465.
[3]	Falkenberg M. Mitochondrial DNA replication in mammalian cells: overview of the pathway. Essays Biochem, 2018, 62(3): 287-296. doi: 10.1042/EBC20170100 pmid: 29880722
[4]	Stewart JB, Chinnery PF. The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease. Nat Rev Genet, 2015, 16(9): 530-542. doi: 10.1038/nrg3966 pmid: 26281784
[5]	Chinnery PF, Thorburn DR, Samuels DC, White SL, Dahl HM, Turnbull DM, Lightowlers RN, Howell N. The inheritance of mitochondrial DNA heteroplasmy: random drift, selection or both? Trends Genet, 2000, 16(11): 500-505. pmid: 11074292
[6]	Gorman GS, Schaefer AM, Ng Y, Gomez N, Blakely EL, Alston CL, Feeney C, Horvath R, Yu-Wai-Man P, Chinnery PF, Taylor RW, Turnbull DM, Mcfarland R. Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease. Ann Neurol, 2015, 77(5): 753-759. doi: 10.1002/ana.24362 pmid: 25652200
[7]	Gorman GS, Chinnery PF, Dimauro S, Hirano M, Koga Y, Mcfarland R, Suomalainen A, Thorburn DR, Zeviani M, Turnbull DM. Mitochondrial diseases. Nat Rev Dis Primers, 2016, 2: 16080. doi: 10.1038/nrdp.2016.80 pmid: 27775730
[8]	Nissanka N, Moraes CT. Mitochondrial DNA heteroplasmy in disease and targeted nuclease-based therapeutic approaches. EMBO Rep, 2020, 21(3): e49612.
[9]	Hyslop LA, Blakeley P, Craven L, Richardson J, Fogarty NME, Fragouli E, Lamb M, Wamaitha SE, Prathalingam N, Zhang Q, O'Keefe H, Takeda Y, Arizzi L, Alfarawati S, Tuppen HA, Irving L, Kalleas D, Choudhary M, Wells D, Murdoch AP, Turnbull DM, Niakan KK, Herbert M. Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease. Nature, 2016, 534(7607): 383-386.
[10]	Tachibana M, Sparman M, Sritanaudomchai H, Ma H, Clepper L, Woodward J, Li Y, Ramsey C, Kolotushkina O, Mitalipov S. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature, 2009, 461(7262): 367-372.
[11]	Srivastava S, Moraes CT. Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease. Hum Mol Genet, 2001, 10(26): 3093-3099. pmid: 11751691
[12]	Minczuk M, Papworth MA, Miller JC, Murphy MP, Klug A. Development of a single-chain, quasi-dimeric zinc- finger nuclease for the selective degradation of mutated human mitochondrial DNA. Nucleic Acids Res, 2008, 36(12): 3926-3938. doi: 10.1093/nar/gkn313 pmid: 18511461
[13]	Bacman SR, Williams SL, Pinto M, Peralta S, Moraes CT. Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs. Nat Med, 2013, 19(9): 1111-1113. doi: 10.1038/nm.3261 pmid: 23913125
[14]	Reddy P, Ocampo A, Suzuki K, Luo JP, Bacman SR, Williams SL, Sugawara A, Okamura D, Tsunekawa Y, Wu J, Lam D, Xiong X, Montserrat N, Esteban CR, Liu GH, Sancho-Martinez I, Manau D, Civico S, Cardellach F, Del Mar O'Callaghan M, Campistol J, Zhao HM, Campistol JM, Moraes CT, Izpisua BJ. Selective elimination of mitochondrial mutations in the germline by genome editing. Cell, 2015, 161(3): 459-469. doi: S0092-8674(15)00371-2 pmid: 25910206
[15]	Gammage PA, Gaude E, Van Haute L, Rebelo-Guiomar P, Jackson CB, Rorbach J, Pekalski ML, Robinson AJ, Charpentier M, Concordet JP, Frezza C, Minczuk M. Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs. Nucleic Acids Res, 2016, 44(16): 7804-7816. doi: 10.1093/nar/gkw676 pmid: 27466392
[16]	Owen RT, Flotte TR. Approaches and limitations to gene therapy for mitochondrial diseases. Antioxid Redox Signal, 2001, 3(3): 451-460.
[17]	Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol, 2011, 29(2): 149-153. doi: 10.1038/nbt.1775 pmid: 21248753
[18]	Mok BY, de Moraes MH, Zeng J, Bosch DE, Kotrys AV, Raguram A, Hsu F, Radey MC, Peterson SB, Mootha VK, Mougous JD, Liu DR. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature, 2020, 583(7817): 631-637.
[19]	Cho SI, Lee S, Mok YG, Lim K, Lee J, Lee JM, Chung E, Kim JS. Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases. Cell, 2022, 185(10): 1764-1776.e12.
[20]	Wei YH, Li ZF, Xu K, Feng H, Xie L, Li D, Zuo ZR, Zhang ML, Xu CL, Yang H, Zuo EW. Mitochondrial base editor DdCBE causes substantial DNA off-target editing in nuclear genome of embryos. Cell Discov, 2022, 8(1): 27. doi: 10.1038/s41421-022-00391-5 pmid: 35304438
[21]	Lei ZX, Meng HW, Liu LL, Zhao HN, Rao XC, Yan YC, Wu H, Liu M, He AB, Yi CQ. Mitochondrial base editor induces substantial nuclear off-target mutations. Nature, 2022, 606(7915): 804-811.
[22]	Mok BY, Kotrys AV, Raguram A, Huang TP, Mootha VK, Liu DR. CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA. Nat Biotechnol, 2022, 40(9): 1378-1387. doi: 10.1038/s41587-022-01256-8 pmid: 35379961
[23]	Mi L, Shi M, Li YX, Xie G, Rao XC, Wu DM, Cheng AM, Niu MX, Xu FL, Yu Y, Gao N, Wei WS, Wang XH, Wang YM. DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing. Nat Commun, 2023, 14(1): 874. doi: 10.1038/s41467-023-36600-2 pmid: 36797253
[24]	Wei YH, Xu CL, Feng H, Xu K, Li ZF, Hu J, Zhou L, Wei Y, Zuo ZR, Zuo EW, Li W, Yang H, Zhang ML. Human cleaving embryos enable efficient mitochondrial base-editing with DdCBE. Cell Discov, 2022, 8(1): 7. doi: 10.1038/s41421-021-00372-0 pmid: 35102133
[25]	Chen XX, Liang D, Guo JY, Zhang JQ, Sun HF, Zhang XL, Jin JC, Dai YC, Bao QM, Qian XZ, Tan L, Hu P, Ling XF, Shen B, Xu ZF. DdCBE-mediated mitochondrial base editing in human 3PN embryos. Cell Discov, 2022, 8(1): 8. doi: 10.1038/s41421-021-00358-y pmid: 35102135
[26]	Wang B, Lv XJ, Wang YF, Wang ZB, Liu Q, Lu B, Liu Y, Gu F. CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome. Sci China Life Sci, 2021, 64(9): 1463-1472. doi: 10.1007/s11427-020-1819-8 pmid: 33420919
[27]	Bi R, Li Y, Xu M, Zheng QZ, Zhang DF, Li X, Ma GL, Xiang BL, Zhu XJ, Zhao H, Huang XX, Zheng P, Yao YG. Direct evidence of CRISPR-Cas9-mediated mitochondrial genome editing. Innovation (Camb), 2022, 3(6): 100329.
[28]	Yi ZY, Zhang XX, Tang W, Yu Y, Wei XX, Zhang X, Wei WS. Strand-selective base editing of human mitochondrial DNA using mitoBEs. Nat Biotechnol, 2023, 42(3): 498-509. doi: 10.1038/s41587-023-01791-y pmid: 37217751

Metrics

Comments

www.chinagene.cn
备案号：京ICP备09063187号

线粒体编辑器	组成元件	编辑类型	偏好性	编辑效率	脱靶	参考文献
CRISPR Cas9	Cas9, gRNA	Indel	碱基识别偏好	极小	严重的脱靶效应	[26,27]
DdCBEs	DddA, TALE	C·G-T·A	偏好TC序列	4.9%~49%	严重的脱靶效应	[18]
DdCBEs(V11)	DddA11, TALE	C·G-T·A	HC	15%~30%	严重的脱靶效应	[22]
DdCBE-Ss	Ddd-Ss, TALE	C·G-T·A	DC	6%~51%	严重的脱靶效应	[23]
Mito-TALED	DddA, TadA8e, TALE	A·T-G·C	编辑位点偏好	大约40%	严重的脱靶效应	[19]
Mito-BEs	TALE, Chymotrypsin, Deaminase	A·T-G·C或 C·G-T·A	链选择偏好	大约40%	暂未发现明确的脱靶效应	[28]

Dual-localization signals enhance mitochondrial targeted presentation of engineered proteins

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 6

References 28

Related Articles 2

Recommended Articles

Metrics

Comments

[1]	Ruijia Song, Lu Han, Haifeng Sun, Bin Shen. Advances in mitochondrial DNA base editing technology [J]. Hereditas(Beijing), 2023, 45(8): 632-642.
[2]	. Mitochondrial DNA mutations and related human dis-eases [J]. HEREDITAS, 2007, 29(11): 1299-1299―1308.