Study of the effect of the introduction of mitochondrial import determinants into the gRNA structure on the activity of the gRNA/SpCas9 complex in vitro

It has long been known that defects in the structure of the mitochondrial genome can cause various neuromuscular and neurodegenerative diseases. Nevertheless, at present there is no effective method for treating mitochondrial diseases. The major problem with the treatment of such diseases is associated with mitochondrial DNA (mtDNA) heteroplasmy. It means that due to a high copy number of the mitochondrial genome, mutant copies of mtDNA coexist with wild-type molecules in the same organelle. The clinical symptoms of mitochondrial diseases and the degree of their manifestation directly depend on the number of mutant mtDNA molecules in the cell. The possible way to reduce adverse effects of the mutation is by shifting the level of heteroplasmy towards the wild-type mtDNA molecules. Using this idea, several gene therapeutic approaches based on TALE and ZF nucleases have been developed for this purpose. However, the construction of protein domains of such systems is rather long and laborious process. Meanwhile, the CRISPR/Cas9 system is fundamentally different from protein systems in that it is easy to use, highly efficiency and has a different mechanism of action. All the characteristics and capabilities of the CRISPR/Cas9 system make it a promising tool in mitochondrial genetic engineering. In this article, we demonstrate for the first time that the modification of gRNA by integration of specific mitochondrial import determinants in the gRNA scaffold does not affect the activity of the gRNA/Cas9 complex in vitro.

1. Anders C., Niewoehner O., Duerst A., Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014;513(7519):569-573. DOI 10.1038/nature13579.

2. Bian W.P., Chen Y.L., Luo J.J., Wang C., Xie S.L., Pei D.S. A knock-in strategy for editing human and zebrafish mitochondrial DNA using mito-CRISPR/Cas9 system. ACS Synth. Biol. 2019;8(4):621-632. DOI 10.1021/acssynbio.8b00411.

3. Briner A.E., Donohoue P.D., Gomaa A.A., Selle K., Slorach E.M., Nye C.H., Haurwitz R.E., Beisel C.L., May A.P., Barrangou R. Guide RNA functional modules direct Cas9 activity and orthogonality. Mol. Cell. 2014;56(2):333-339. DOI 10.1016/j.molcel.2014.09.019.

4. Calvo S.E., Mootha V.K. The mitochondrial proteome and human disease. Annu. Rev. Genomics Hum. Genet. 2010;11(1):25-44. DOI 10.1146/annurev-genom-082509-141720.

5. Chang D.D., Clayton D.A. A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA. Science. 1987;235: 1178-1184. DOI 10.1126/science.2434997.

6. Comte C., Tonin Y., Heckel-Mager A.-M., Boucheham A., Smirnov A., Auré K., Lombès A., Martin R.P., Entelis N., Tarassov I. Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome. Nucleic Acids Res. 2013;41(1):418-433. DOI 10.1093/nar/gks965.

7. Doersen C.J., Guerrier-Takada C., Altman S., Attardi G. Characterization of an RNase P activity from HeLa cell mitochondria. Comparison with the cytosol RNase P activity. J. Biol. Chem. 1985; 260(10): 5942-5949.

8. Fan S., Tian T., Chen W., Lv X., Lei X., Zhang H., Sun S., Cai L., Pan G., He L., Ou Z., Lin X., Wang X., Perez M.F., Tu Z., Ferrone S., Tannous B.A., Li J. Mitochondrial miRNA determines chemoresistance by reprogramming metabolism and regulating mitochondrial transcription. Cancer Res. 2019:79(6):1069-1084. DOI 10.1158/0008-5472.CAN-18-2505.

9. Gammage P.A., Moraes C.T., Minczuk M. Mitochondrial genome engineering: the revolution may not be CRISPR-Ized. Trends Genet. 2018;34(2):101-110. DOI 10.1016/j.tig.2017.11.001.

10. Gowher A., Smirnov A., Tarassov I., Entelis N. Induced tRNA import into human mitochondria: implication of a host aminoacyl-tRNAsynthetase. PLoS One. 2013;8(6):e66228. DOI 10.1371/journal.pone.0066228.

11. Holzmann J., Frank P., Löffler E., Bennett K.L., Gerner C., Rossmanith W. RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme. Cell. 2008;135(3):462-474. DOI 10.1016/j.cell.2008.09.013.

12. Jeandard D., Smirnova A., Tarassov I., Barrey E., Smirnov A., Entelis N. Import of non-coding RNAs into human mitochondria: A critical review and emerging approaches. Cells. 2019;8(3):286. DOI 10.3390/cells8030286.

13. Jiang F., Doudna J.A. CRISPR-Cas9 structures and mechanisms. Annu. Rev. Biophys. 2017;46(1):505-529. DOI 10.1146/annurevbiophys-062215-010822.

14. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821. DOI 10.1126/science.1225829.

15. Jinek M., Jiang F., Taylor D.W., Sternberg S.H., Kaya E., Ma E., Anders C., Hauer M., Zhou K., Lin S., Kaplan M., Iavarone A.T., Charpentier E., Nogales E., Doudna J.A. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science. 2014;343(6176):1247997. DOI 10.1126/science.1247997.

16. Jo A., Ham S., Lee G.H., Lee Y.I., Kim S., Lee Y.S., Shin J.H., Lee Y. Efficient mitochondrial genome editing by CRISPR/Cas9. BioMed Res. Int. 2015;2015:305716. DOI 10.1155/2015/305716.

17. Kamenski P., Kolesnikova O., Jubenot V., Entelis N., Krasheninnikov I.A., Martin R.P., Tarassov I. Evidence for an adaptation mechanism of mitochondrial translation via tRNA import from the cytosol. Mol. Cell. 2007;26(5):625-637. DOI 10.1016/j.molcel.2007.04.019.

18. Kamenski P., Smirnova E., Kolesnikova O., Krasheninnikov I.A., Martin R.P., Entelis N., Tarassov I. tRNA mitochondrial import in yeast: Mapping of the import determinants in the carrier protein, the precursor of mitochondrial lysyl-tRNA synthetase. Mitochondrion. 2010;10(3):284-293. DOI 10.1016/j.mito.2010.01.002.

19. Kazakova H.A., Entelis N.S., Martin R.P., Tarassov I.A. The aminoacceptor stem of the yeast tRNA(Lys) contains determinants of mitochondrial import selectivity. FEBS Lett. 1999;442(2-3):193-197. DOI 10.1016/S0014-5793(98)01653-6.

20. Komor A.C., Badran A.H., Liu D.R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell. 2017;168(1-2):20-36. DOI 10.1016/j.cell.2016.10.044.

21. Konermann S., Brigham M.D., Trevino A.E., Joung J., Abudayyeh O.O., Barcena C., Hsu P.D., Habib N., Gootenberg J.S., Nishimasu H., Nureki O., Zhang F. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 2015;517(7536):583-588. DOI 10.1038/nature14136.

22. Lorenz R., Bernhart S.H., Höner zu Siederdissen C., Tafer H., Flamm C., Stadler P.F., Hofacker I.L. ViennaRNA Package 2.0. Algorithms Mol. Biol. 2011;6(1):26. DOI 10.1186/1748-7188-6-26.

23. Loutre R., Heckel A.M., Smirnova A., Entelis N., Tarassov I. Can mitochondrial DNA be CRISPRized: Pro and contra. IUBMB Life. 2018; 70(12):1233-1239. DOI 10.1002/iub.1919.

24. Ma H., Tu L.C., Naseri A., Huisman M., Zhang S., Grunwald D., Pederson T. Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat. Biotechnol. 2016; 34(5):528-530. DOI 10.1038/nbt.3526.

25. Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., Norville J.E., Church G.M. RNA-guided human genome engineering via Cas9. Science. 2013;339/6121:823-826. DOI 10.1126/science.1232033.

26. Markantone D.M., Towheed A., Crain A.T., Collins J.M., Celotto A.M., Palladino M.J. Protein coding mitochondrial-targeted RNAs rescue mitochondrial disease in vivo. Neurobiol. Dis. 2018;117:203-210. DOI 10.1016/j.nbd.2018.06.009.

27. Martin R.P., Schneller J.M., Stahl A.J., Dirheimer G. Import of nuclear deoxyribonucleic acid coded lysine-accepting transfer ribonucleic acid (anticodon C-U-U) into yeast mitochondria. Biochemistry. 1979;18(21):4600-4605. DOI 10.1021/bi00588a021.

28. Nishimasu H., Ran F.A., Hsu P.D., Konermann S., Shehata S.I., Dohmae N., Ishitani R., Zhang F., Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156(5):935- 949. DOI 10.1016/j.cell.2014.02.001.

29. Noh J.H., Kim K.M., Abdelmohsen K., Yoon J.H., Panda A.C., Munk R., Kim J., Curtis J., Moad C.A., Wohler C.M., Indig F.E., de Paula W., Dudekula D.B., De S., Piao Y., Yang X., Martindale J.L., de Cabo R., Gorospe M. HuR and GRSF1 modulate the nuclear export and mitochondrial localization of the lncRNA RMRP. Genes Dev. 2016;30(10):1224-1239. DOI 10.1101/gad.276022.115.

30. Nowak C.M., Lawson S., Zerez M., Bleris L. Guide RNA engineering for versatile Cas9 functionality. Nucleic Acids Res. 2016;44(20): 9555-9564. DOI 10.1093/nar/gkw908.

31. Orishchenko К.Е., Sofronova J.К., Chupakhin Е.G., Lunev E.A., Маzunin I.О. Delivery Cas9 into mitochondria. Geny i Kletki = Genes and Cells. 2016;11(2):100-105. (in Russisn)

32. Pfanner N., Warscheid B., Wiedemann N. Mitochondrial proteins: from biogenesis to functional networks. Nat. Rev. Mol. Cell Biol. 2019;20:267-284. DOI 10.1038/s41580-018-0092-0.

33. Ran F.A., Hsu P.D., Wright J., Agarwala V., Scott D.A., Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013;8:2281-2308. DOI 10.1038/nprot.2013.143.

34. Rubio M.A., Rinehart J.J., Krett B., Duvezin-Caubet S., Reichert A.S., Söll D., Alfonzo J.D. Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import. Proc. Natl. Acad. Sci. USA. 2008;105(27):9186-9191. DOI 10.1073/pnas.0804283105.

35. Shao S., Zhang W., Hu H., Xue B., Qin J., Sun C., Sun Y., Wei W., Sun Y. Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system. Nucleic Acids Res. 2016;44(9):e86- e86. DOI 10.1093/nar/gkw066.

36. Tonin Y., Heckel A.M., Vysokikh M., Dovydenko I., Meschaninova M., Rötig A., Munnich A., Venyaminova A., Tarassov I., Entelis N. Modeling of antigenomic therapy of mitochondrial diseases by mitochondrially addressed RNA targeting a pathogenic point mutation in mitochondrial DNA. J. Biol. Chem. 2014;289(19):13323-13334. DOI 10.1074/jbc.M113.528968.

37. Verechshagina N., Nikitchina N., Yamada Y., Harashima H., Tanaka M., Orishchenko K., Mazunin I. Future of human mitochondrial DNA editing technologies. Mitochondrial DNA A DNA Mapp Seq. Anal. 2019;30(2):214-221. DOI 10.1080/24701394.2018.1472773.

38. Wang G., Chen H.-W.W., Oktay Y., Zhang J., Allen E.L., Smith G.M., Fan K.C., Hong J.S., French S.W., McCaffery J.M., Lightowlers R.N., Morse H.C., Koehler C.M., Teitell M.A. PNPASE regulates RNA import into mitochondria. Cell. 2010;142(3):456-467. DOI 10.1016/j.cell.2010.06.035.

39. Wang G., Shimada E., Zhang J., Hong J.S., Smith G.M., Teitell M.A., Koehler C.M. Correcting human mitochondrial mutations with targeted RNA import. Proc. Natl. Acad. Sci. USA. 2012;109(13):4840- 4845. DOI 10.1073/pnas.1116792109.

40. Wright A.V., Sternberg S.H., Taylor D.W., Staahl B.T., Bardales J.A., Kornfeld J.E., Doudna J.A. Rational design of a split-Cas9 enzyme complex. Proc. Natl. Acad. Sci. USA. 2015;112(10):2984-2989. DOI 10.1073/pnas.1501698112.

41. Zalatan J.G., Lee M.E., Almeida R., Gilbert L.A., Whitehead E.H., La Russa M., Tsai J.C., Weissman J.S., Dueber J.E., Qi L.S., Lim W.A. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell. 2015;160(1-2):339-350. DOI 10.1016/j.cell.2014.11.052.