Publications 2019 – 2020

1. Sasnauskas G, Siksnys V. CRISPR adaptation from a structural perspective. Curr Opin Struct Biol. 2020 Dec;65:17-25. doi: 10.1016/j.sbi.2020.05.015. Epub 2020 Jun 20.
PMID: 32570107

2. Songailiene I, Juozapaitis J, Tamulaitiene G, Ruksenaite A, Šulčius S, Sasnauskas G, Venclovas Č, Siksnys V. HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation. Mol Cell. 2020 Dec 17;80(6):955-970.e7. doi: 10.1016/j.molcel.2020.11.034. Epub 2020 Dec 7.
PMID: 33290744

3. Gasiunas G, Young JK, Karvelis T, Kazlauskas D, Urbaitis T, Jasnauskaite M, Grusyte MM, Paulraj S, Wang PH, Hou Z, Dooley SK, Cigan M, Alarcon C, Chilcoat ND, Bigelyte G, Curcuru JL, Mabuchi M, Sun Z, Fuchs RT, Schildkraut E, Weigele PR, Jack WE, Robb GB, Venclovas Č, Siksnys V. A catalogue of biochemically diverse CRISPR-Cas9 orthologs. Nat Commun. 2020 Nov 2;11(1):5512. doi: 10.1038/s41467-020-19344-1.
PMID: 33139742

4. Makarova KS, Timinskas A, Wolf YI, Gussow AB, Siksnys V, Venclovas Č, Koonin EV. Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense. Nucleic Acids Res. 2020 Sep 18;48(16):8828-8847. doi: 10.1093/nar/gkaa635.
PMID: 32735657

5. Smalakyte D, Kazlauskiene M, F Havelund J, Rukšėnaitė A, Rimaite A, Tamulaitiene G, Færgeman NJ, Tamulaitis G, Siksnys V. Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains. Nucleic Acids Res. 2020 Sep 18;48(16):9204-9217. doi: 10.1093/nar/gkaa634.
PMID: 32766806

6. Fricke T, Smalakyte D, Lapinski M, Pateria A, Weige C, Pastor M, Kolano A, Winata C, Siksnys V, Tamulaitis G, Bochtler M. Targeted RNA Knockdown by a Type III CRISPR-Cas Complex in Zebrafish.
CRISPR J. 2020 Aug;3(4):299-313. doi: 10.1089/crispr.2020.0032.
PMID: 32833532

7. Mullally G, van Aelst K, Naqvi MM, Diffin FM, Karvelis T, Gasiunas G, Siksnys V, Szczelkun MD. 5' modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage.
Nucleic Acids Res. 2020 Jul 9;48(12):6811-6823. doi: 10.1093/nar/gkaa477.
PMID: 32496535

8. Karvelis T, Bigelyte G, Young JK, Hou Z, Zedaveinyte R, Budre K, Paulraj S, Djukanovic V, Gasior S, Silanskas A, Venclovas Č, Siksnys V. PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Res. 2020 May 21;48(9):5016-5023. doi: 10.1093/nar/gkaa208.
PMID: 32246713

9. Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, Charpentier E, Cheng D, Haft DH, Horvath P, Moineau S, Mojica FJM, Scott D, Shah SA, Siksnys V, Terns MP, Venclovas Č, White MF, Yakunin AF, Yan W, Zhang F, Garrett RA, Backofen R, van der Oost J, Barrangou R, Koonin EV. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020 Feb;18(2):67-83. doi: 10.1038/s41579-019-0299-x. Epub 2019 Dec 19.
PMID: 31857715

10. Karvelis T, Young JK, Siksnys V. A pipeline for characterization of novel Cas9 orthologs. Methods Enzymol. 2019;616:219-240. doi: 10.1016/bs.mie.2018.10.021. Epub 2018 Dec 27.
PMID: 30691644

11. Songailiene I, Rutkauskas M, Sinkunas T, Manakova E, Wittig S, Schmidt C, Siksnys V, Seidel R. Decision-Making in Cascade Complexes Harboring crRNAs of Altered Length. Cell Rep. 2019 Sep 17;28(12):3157-3166.e4. doi: 10.1016/j.celrep.2019.08.033.
PMID: 31533038

12. Wilkinson M, Drabavicius G, Silanskas A, Gasiunas G, Siksnys V, Wigley DB. Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System. Mol Cell. 2019 Jul 11;75(1):90-101.e5. doi: 10.1016/j.molcel.2019.04.020. Epub 2019 May 9.
PMID: 31080012

13. Young J, Zastrow-Hayes G, Deschamps S, Svitashev S, Zaremba M, Acharya A, Paulraj S, Peterson-Burch B, Schwartz C, Djukanovic V, Lenderts B, Feigenbutz L, Wang L, Alarcon C, Siksnys V, May G, Chilcoat ND, Kumar S. CRISPR-Cas9 Editing in Maize: Systematic Evaluation of Off-target Activity and Its Relevance in Crop Improvement. Sci Rep. 2019 Apr 30;9(1):6729. doi: 10.1038/s41598-019-43141-6.
PMID: 31040331

14. Mogila I, Kazlauskiene M, Valinskyte S, Tamulaitiene G, Tamulaitis G, Siksnys V. Genetic Dissection of the Type III-A CRISPR-Cas System Csm Complex Reveals Roles of Individual Subunits. Cell Rep. 2019 Mar 5;26(10):2753-2765.e4. doi: 10.1016/j.celrep.2019.02.029.
PMID: 30840895

15. Tamulaitiene G, Manakova E, Jovaisaite V, Tamulaitis G, Grazulis S, Bochtler M, Siksnys V. Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family. Nucleic Acids Res. 2019 Jan 25;47(2):997-1010. doi: 10.1093/nar/gky1137.
PMID: 30445642

16. Gordeeva J, Morozova N, Sierro N, Isaev A, Sinkunas T, Tsvetkova K, Matlashov M, Truncaite L, Morgan RD, Ivanov NV, Siksnys V, Zeng L, Severinov K. BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site. Nucleic Acids Res. 2019 Jan 10;47(1):253-265. doi: 10.1093/nar/gky1125.
PMID: 30418590

1. Sasnauskas G, Siksnys V. CRISPR adaptation from a structural perspective. Curr Opin Struct Biol. 2020 Dec;65:17-25. doi: 10.1016/j.sbi.2020.05.015. Epub 2020 Jun 20.
PMID: 32570107

2. Songailiene I, Juozapaitis J, Tamulaitiene G, Ruksenaite A, Šulčius S, Sasnauskas G, Venclovas Č, Siksnys V. HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation. Mol Cell. 2020 Dec 17;80(6):955-970.e7. doi: 10.1016/j.molcel.2020.11.034. Epub 2020 Dec 7.
PMID: 33290744

3. Gasiunas G, Young JK, Karvelis T, Kazlauskas D, Urbaitis T, Jasnauskaite M, Grusyte MM, Paulraj S, Wang PH, Hou Z, Dooley SK, Cigan M, Alarcon C, Chilcoat ND, Bigelyte G, Curcuru JL, Mabuchi M, Sun Z, Fuchs RT, Schildkraut E, Weigele PR, Jack WE, Robb GB, Venclovas Č, Siksnys V. A catalogue of biochemically diverse CRISPR-Cas9 orthologs. Nat Commun. 2020 Nov 2;11(1):5512. doi: 10.1038/s41467-020-19344-1.
PMID: 33139742

4. Makarova KS, Timinskas A, Wolf YI, Gussow AB, Siksnys V, Venclovas Č, Koonin EV. Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense. Nucleic Acids Res. 2020 Sep 18;48(16):8828-8847. doi: 10.1093/nar/gkaa635.
PMID: 32735657

5. Smalakyte D, Kazlauskiene M, F Havelund J, Rukšėnaitė A, Rimaite A, Tamulaitiene G, Færgeman NJ, Tamulaitis G, Siksnys V. Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains. Nucleic Acids Res. 2020 Sep 18;48(16):9204-9217. doi: 10.1093/nar/gkaa634.
PMID: 32766806

6. Fricke T, Smalakyte D, Lapinski M, Pateria A, Weige C, Pastor M, Kolano A, Winata C, Siksnys V, Tamulaitis G, Bochtler M. Targeted RNA Knockdown by a Type III CRISPR-Cas Complex in Zebrafish.
CRISPR J. 2020 Aug;3(4):299-313. doi: 10.1089/crispr.2020.0032.
PMID: 32833532

7. Mullally G, van Aelst K, Naqvi MM, Diffin FM, Karvelis T, Gasiunas G, Siksnys V, Szczelkun MD. 5' modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage.
Nucleic Acids Res. 2020 Jul 9;48(12):6811-6823. doi: 10.1093/nar/gkaa477.
PMID: 32496535

8. Karvelis T, Bigelyte G, Young JK, Hou Z, Zedaveinyte R, Budre K, Paulraj S, Djukanovic V, Gasior S, Silanskas A, Venclovas Č, Siksnys V. PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Res. 2020 May 21;48(9):5016-5023. doi: 10.1093/nar/gkaa208.
PMID: 32246713

9. Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, Charpentier E, Cheng D, Haft DH, Horvath P, Moineau S, Mojica FJM, Scott D, Shah SA, Siksnys V, Terns MP, Venclovas Č, White MF, Yakunin AF, Yan W, Zhang F, Garrett RA, Backofen R, van der Oost J, Barrangou R, Koonin EV. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020 Feb;18(2):67-83. doi: 10.1038/s41579-019-0299-x. Epub 2019 Dec 19.
PMID: 31857715

10. Karvelis T, Young JK, Siksnys V. A pipeline for characterization of novel Cas9 orthologs. Methods Enzymol. 2019;616:219-240. doi: 10.1016/bs.mie.2018.10.021. Epub 2018 Dec 27.
PMID: 30691644

11. Songailiene I, Rutkauskas M, Sinkunas T, Manakova E, Wittig S, Schmidt C, Siksnys V, Seidel R. Decision-Making in Cascade Complexes Harboring crRNAs of Altered Length. Cell Rep. 2019 Sep 17;28(12):3157-3166.e4. doi: 10.1016/j.celrep.2019.08.033.
PMID: 31533038

12. Wilkinson M, Drabavicius G, Silanskas A, Gasiunas G, Siksnys V, Wigley DB. Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System. Mol Cell. 2019 Jul 11;75(1):90-101.e5. doi: 10.1016/j.molcel.2019.04.020. Epub 2019 May 9.
PMID: 31080012

13. Young J, Zastrow-Hayes G, Deschamps S, Svitashev S, Zaremba M, Acharya A, Paulraj S, Peterson-Burch B, Schwartz C, Djukanovic V, Lenderts B, Feigenbutz L, Wang L, Alarcon C, Siksnys V, May G, Chilcoat ND, Kumar S. CRISPR-Cas9 Editing in Maize: Systematic Evaluation of Off-target Activity and Its Relevance in Crop Improvement. Sci Rep. 2019 Apr 30;9(1):6729. doi: 10.1038/s41598-019-43141-6.
PMID: 31040331

14. Mogila I, Kazlauskiene M, Valinskyte S, Tamulaitiene G, Tamulaitis G, Siksnys V. Genetic Dissection of the Type III-A CRISPR-Cas System Csm Complex Reveals Roles of Individual Subunits. Cell Rep. 2019 Mar 5;26(10):2753-2765.e4. doi: 10.1016/j.celrep.2019.02.029.
PMID: 30840895

15. Tamulaitiene G, Manakova E, Jovaisaite V, Tamulaitis G, Grazulis S, Bochtler M, Siksnys V. Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family. Nucleic Acids Res. 2019 Jan 25;47(2):997-1010. doi: 10.1093/nar/gky1137.
PMID: 30445642

16. Gordeeva J, Morozova N, Sierro N, Isaev A, Sinkunas T, Tsvetkova K, Matlashov M, Truncaite L, Morgan RD, Ivanov NV, Siksnys V, Zeng L, Severinov K. BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site. Nucleic Acids Res. 2019 Jan 10;47(1):253-265. doi: 10.1093/nar/gky1125.
PMID: 30418590

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Publikacijos 2017 – 2018

  1. Czapinska, H., Kowalska, M., Zagorskaitė, E., Manakova, E., Slyvka, A., Xu, S.-y., Siksnys, V., Sasnauskas, G. & Bochtler, M., Activity and structure of EcoKMcrA Nucleic Acids Research, 2018. (PDF)
  2. Drabavicius, G., Sinkunas, T., Silanskas, A., Gasiunas, G., Venclovas, Č. & Siksnys, V., DnaQ exonuclease‐like domain of Cas2 promotes spacer integration in a type I‐E CRISPR‐Cas system EMBO reports, 2018, vol. 19, e45543. (PDF)
  3. Gordeeva, J., Morozova, N., Sierro, N., Isaev, A., Sinkunas, T., Tsvetkova, K., Matlashov, M., Truncaite, L., Morgan, R. D., Ivanov, N. V., Siksnys, V., Zeng, L. & Severinov, K., {BREX} system of {E}scherichia coli distinguishes self from non-self by methylation of a specific {DNA} site Nucleic Acids Research, 2018. (PDF)
  4. Sasnauskas, G., Manakova, E., Lapėnas, K., Kauneckaitė, K. & Siksnys, V., DNA recognition by Arabidopsis transcription factors ABI3 and NGA1 The FEBS Journal, 2018, vol. 285, 4041–4059. (PDF)
  5. Sasnauskas, G., Kauneckaitė, K. & Siksnys, V., Structural basis of DNA target recognition by the B3 domain of Arabidopsis epigenome reader VAL1 Nucleic Acids Research, 2018, vol. 46, 4316–4324. (PDF)
  6. Tamulaitiene, G., Manakova, E., Jovaisaite, V., Tamulaitis, G., Grazulis, S., Bochtler, M. & Siksnys, V., Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family Nucleic Acids Research, 2018. (PDF)
  7. Toliusis, P., Tamulaitiene, G., Grigaitis, R., Tuminauskaite, D., Silanskas, A., Manakova, E., Venclovas, Č., Szczelkun, M. D., Siksnys, V. & Zaremba, M., The H-subunit of the restriction endonuclease CglI contains a prototype DEAD-Z1 helicase-like motor Nucleic Acids Research, 2018, vol. 46, 2560–2572. (PDF)
  8. Karvelis, T., Gasiunas, G. & Siksnys, V., Harnessing the natural diversity and in vitro evolution of Cas9 to expand the genome editing toolbox Current Opinion in Microbiology, 2017, vol. 37, 88–94. (PDF)
  9. Karvelis, T., Gasiunas, G. & Siksnys, V., Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: A brief overview Methods, 2017, vol. 121-122, 3–8. (PDF)
  10. Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G. & Siksnys, V., A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems Science, 2017, vol. 357, 605–609. (PDF)
  11. Kostiuk, G., Dikić, J., Schwarz, F. W., Sasnauskas, G., Seidel, R. & Siksnys, V., The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA Nucleic Acids Research, 2017, vol. 45, 5968–5979. (PDF)
  12. Sasnauskas, G., Tamulaitienė, G., Tamulaitis, G., Čalyševa, J., Laime, M., Rimšelienė, R., Lubys, A. & Siksnys, V., UbaLAI is a monomeric Type IIE restriction enzyme Nucleic Acids Research, 2017, vol. 45, 9583–9594. (PDF)
  13. Toliusis, P., Zaremba, M., Silanskas, A., Szczelkun, M. D. & Siksnys, V., CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases Nucleic Acids Research, 2017, vol. 45, 8435–8447. (PDF)
  14. Tamulaitiene, G., Jovaisaite, V., Tamulaitis, G., Songailiene, I., Manakova, E., Zaremba, M., Grazulis, S., Xu, S.-y. & Siksnys, V., Restriction endonuclease AgeI is a monomer which dimerizes to cleave DNA Nucleic Acids Research, 2016, gkw1310. (PDF)

Restrikcijos endonukleazės

Restrikcijos endonukleazių struktūros ir veikimo mechanizmų tyrimai

Restrikcijos ir modifikacijos (RM) sistemos yra vienas iš ginklų, kurį bakterijos ląstelė naudoja apsaugai nuo svetimos DNR ir bakteriofagų antpuolio. RM sistemos paprastai susideda iš dviejų fermentų: restrikcijos endonukleazės (REazės) bei metiltransferazės (MTazės). REazė karpo tik "svetimą" DNR, bet neliečia "savos", nes greta esanti MTazė pažymi "savo" DNR įvesdama į REazės taikinį metilo grupę. Šiuo metu yra aprašyta virš 330 skirtingų specifiškumų REazių iš 4000 bakterijų rūšių. Šie fermentai yra nepakeičiami įrankiai laboratorijoje, nes veikia kaip "molekulinės žirklės", kurios karpo DNR. Įdomu, kad tą pačią funkciją atliekančios REazės pasižymi didele tiek struktūrų, tiek DNR kirpimo mechanizmų įvairove. Svarbiausi klausimai, į kuriuos mes norime rasti atsakymus tirdami REazes Baltymų-nukleorūgščių sąveikos tyrimų skyriuje yra šie:

  • Kaip restrikcijos fermentai atpažįsta "savo" specifines sekas?
  • Kokie bendri struktūriniai ir molekuliniai mechanizmai sieja restrikcijos fermentus, atpažįstančius giminingas DNR sekas?
  • Kaip DNR atpažinimas yra susijęs su katalize?
  • Ar galima sukurti naujus restrikcijos fermentus, supratus jų sandarą ir veikimo principus?

Atsakymų į šiuos klausimus mes ieškome pasitelkdami baltymų-DNR kompleksą rentgenostruktūrinę analizę, kryptingą mutagenezę ir biocheminius metodus.