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Research Article

volume 54 issue 9 (september 2020) : 1086-1097,   Doi: 10.18805/ijar.B-3880

Exploration of RNA-Guided Recombinase Target Sites for Hyperactivated Recombinase Beta in Bovine Genome 

S
Shalu Kumari Pathak
A
Arvind Sonwane
S
Subodh Kumar
1ICAR- Indian Veterinary Research Institute, Animal Genetics, Bareilly-243 122, Uttar Pradesh, India. 
Cite article:- Pathak Kumari Shalu, Sonwane Arvind, Kumar Subodh (2019). Exploration of RNA-Guided Recombinase Target Sites for Hyperactivated Recombinase Beta in Bovine Genome. Indian Journal of Animal Research. 54(9): 1086-1097. doi: 10.18805/ijar.B-3880.

ABSTRACT

RNA-guided recombinases (RGR) are potentially valuable tools for basic research and genetic modifications. The platform has been demonstrated to do genome editing efficiently. The platform operates on a typical recognition site comprised of the degenerate recombinase site, a 5 to 6-base pair spacer flanking it and this whole central region is flanked by two guide RNA-specified DNA sequences or Cas9 binding sites which is followed by protospacer adjacent motifs. In present investigation, a detailed map of target sites for RNA-guided recombinase platforms based on hyperactivated recombinase Beta throughout the bovine genome was prepared. For this, Chromosome wise whole genomic sequence data was retrieved from Ensembl followed by designing search pattern for recombinase Beta with spacer length five. By using this search pattern, RGR target sites were located by using dreg program of Emboss package. In total,436 RGR target sites were identified in bovine genome for recombinase Beta with spacer length five. These RGR target site provide potential of being utilized for specific genomic integration, deletion or inversion.

REFERENCES

  1. Akopian, A., He, J., Boocock, M. R. and Stark, W. M. (2003). Chimeric recombinases with designed DNA sequence recognition. Proceedings of the National Academy of Sciences of the United States of America. 100: 8688-8691.
  2. Chaikind, B., Bessen L. J., Thompson, D. B., Hu, J. H. and Liu, R. D. (2016). A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucleic Acids Research. 44: 9758-9770.
  3. Chalberg,T.W., Portlock,J.L., Olivares, E.C., Thyagarajan,B., Kirby,P.J., Hillman,R.T., Hoelters, J. and Calos,M.P. (2006). Integration specificity of phage phiC31 integrase in the human genome. Journal of Moecular Biology. 357: 28-48.
  4. Crooks, G.E., Hon, G., Chandonia, J.M. and Brenner, S.E. (2004). WebLogo: a sequence logo generator. Genome Research. 14(6): 1188–90. 
  5. Cunningham F., Amode M.R., Barrell D., Beal K., Billis K., Brent S., Carvalho-Silva D., Clapham P., Coates G., Fitzgerald S., et al. (2015). Nucleic Acids Research. 43: D662–D669.
  6. Gaj, T., Mercer, A. C., Gersbach, C. A., Gordley, R. M. and Barbas, C. F. 3rd. (2011). Structure-guided reprogramming of serine recombinase DNA sequence specificity. Proceedings of the National Academy of Sciences of the United States of America. 108: 498–503.
  7. Gaj, T., Mercer, A. C., Sirk, S. J., Smith, H. L. and Barbas, C. F. 3rd. (2013). A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic Acids Research. 41: 3937-3946.
  8. Gaj T., Sirk, S.J. and Barbas, C.F. III (2014). Expanding the scope of site-specific recombinases for genetic and metabolic engineering. Biotechnology and Bioengineering. 111: 1–15.
  9. Gentleman R.C., Carey V.J., Bates D.M., Bolstad B., Dettling M., Dudoit S., Ellis B., Gautier L., Ge Y., Gentry J., et al. (2004). Bioconductor: Open software development for computational biology and bioinformatics. Genome Biology. 5: R80.
  10. Gersbach, C. A., Gaj, T., Gordley, R. M., Mercer, A. C. and Barbas, C. F. 3rd. (2011). Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase. Nucleic Acids Research. 39: 7868–7878.
  11. Gordley, R. M., Gersbach, C. A. and Barbas, C. F. 3rd. (2009). Synthesis of programmable integrases. Proceedings of the National Academy of Sciences of the United States of America. 106: 5053-5058.
  12. Grainge, I. and Jayaram, M. (1999). The integrase family of recombinase: Organization and function of the active site. Molecular Microbiology. 33(3): 449–456.
  13. Grindley,N.D.F., Whiteson,K.L. and Rice,P.A. (2006). Mechanisms of site-specific recombination. Annual Review of Biochemistry. 75: 567–605.
  14. Mercer, A. C., Gaj, T., Fuller, R. P. and Barbas, C. F. (2012). Chimeric TALE recombinases with programmable DNA sequence specificity. Nucleic Acids Research. 40: 11163–11172.
  15. Prorocic, M. M., Wenlong, D., Olorunniji, F. J., Akopian, A., Schloetel, J. G., Hannigan, A., McPherson, A. L. and Stark, W. M. (2011). Zinc-finger recombinase activities in vitro. Nucleic Acids Research. 39: 9316–9328.
  16. Sancar,A., Lindsey-Boltz,L.A., Unsal-Kacmaz,K. and Linn,S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual Review of Biochemistry. 73: 39–85.
  17. Sarkar, I., Hauber, I., Hauber, J. and Buchholz, F. (2007). HIV-1 proviral DNA excision using an evolved recombinase. Science. 316: 1912-1915.
  18. Sirk, S. J., Gaj, T., Jonsson, A., Mercer, A. C. and Barbas, C. F. (2014). Expanding the zinc-finger recombinase repertoire: Directed evolution and mutational analysis of serine recombinase specificity determinants. Nucleic Acids Research. 42: 4755–4766.
  19. Smith,M.C. and Thorpe,H.M. (2002). Diversity in the serine recombinases. Mol. Microbiol. 44: 299–307.
  20. Thyagarajan,B., Guimaraes,M.J., Groth,A.C. and Calos,M.P. (2000). Mammalian genomes contain active recombinase recognition sites. Gene. 244: 47–54. 
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Indian Journal of Animal Research
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    Research Article

    volume 54 issue 9 (september 2020) : 1086-1097,   Doi: 10.18805/ijar.B-3880

    Exploration of RNA-Guided Recombinase Target Sites for Hyperactivated Recombinase Beta in Bovine Genome 

    S
    Shalu Kumari Pathak
    A
    Arvind Sonwane
    S
    Subodh Kumar
    1ICAR- Indian Veterinary Research Institute, Animal Genetics, Bareilly-243 122, Uttar Pradesh, India. 
    Cite article:- Pathak Kumari Shalu, Sonwane Arvind, Kumar Subodh (2019). Exploration of RNA-Guided Recombinase Target Sites for Hyperactivated Recombinase Beta in Bovine Genome. Indian Journal of Animal Research. 54(9): 1086-1097. doi: 10.18805/ijar.B-3880.

    ABSTRACT

    RNA-guided recombinases (RGR) are potentially valuable tools for basic research and genetic modifications. The platform has been demonstrated to do genome editing efficiently. The platform operates on a typical recognition site comprised of the degenerate recombinase site, a 5 to 6-base pair spacer flanking it and this whole central region is flanked by two guide RNA-specified DNA sequences or Cas9 binding sites which is followed by protospacer adjacent motifs. In present investigation, a detailed map of target sites for RNA-guided recombinase platforms based on hyperactivated recombinase Beta throughout the bovine genome was prepared. For this, Chromosome wise whole genomic sequence data was retrieved from Ensembl followed by designing search pattern for recombinase Beta with spacer length five. By using this search pattern, RGR target sites were located by using dreg program of Emboss package. In total,436 RGR target sites were identified in bovine genome for recombinase Beta with spacer length five. These RGR target site provide potential of being utilized for specific genomic integration, deletion or inversion.

    REFERENCES

    1. Akopian, A., He, J., Boocock, M. R. and Stark, W. M. (2003). Chimeric recombinases with designed DNA sequence recognition. Proceedings of the National Academy of Sciences of the United States of America. 100: 8688-8691.
    2. Chaikind, B., Bessen L. J., Thompson, D. B., Hu, J. H. and Liu, R. D. (2016). A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucleic Acids Research. 44: 9758-9770.
    3. Chalberg,T.W., Portlock,J.L., Olivares, E.C., Thyagarajan,B., Kirby,P.J., Hillman,R.T., Hoelters, J. and Calos,M.P. (2006). Integration specificity of phage phiC31 integrase in the human genome. Journal of Moecular Biology. 357: 28-48.
    4. Crooks, G.E., Hon, G., Chandonia, J.M. and Brenner, S.E. (2004). WebLogo: a sequence logo generator. Genome Research. 14(6): 1188–90. 
    5. Cunningham F., Amode M.R., Barrell D., Beal K., Billis K., Brent S., Carvalho-Silva D., Clapham P., Coates G., Fitzgerald S., et al. (2015). Nucleic Acids Research. 43: D662–D669.
    6. Gaj, T., Mercer, A. C., Gersbach, C. A., Gordley, R. M. and Barbas, C. F. 3rd. (2011). Structure-guided reprogramming of serine recombinase DNA sequence specificity. Proceedings of the National Academy of Sciences of the United States of America. 108: 498–503.
    7. Gaj, T., Mercer, A. C., Sirk, S. J., Smith, H. L. and Barbas, C. F. 3rd. (2013). A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic Acids Research. 41: 3937-3946.
    8. Gaj T., Sirk, S.J. and Barbas, C.F. III (2014). Expanding the scope of site-specific recombinases for genetic and metabolic engineering. Biotechnology and Bioengineering. 111: 1–15.
    9. Gentleman R.C., Carey V.J., Bates D.M., Bolstad B., Dettling M., Dudoit S., Ellis B., Gautier L., Ge Y., Gentry J., et al. (2004). Bioconductor: Open software development for computational biology and bioinformatics. Genome Biology. 5: R80.
    10. Gersbach, C. A., Gaj, T., Gordley, R. M., Mercer, A. C. and Barbas, C. F. 3rd. (2011). Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase. Nucleic Acids Research. 39: 7868–7878.
    11. Gordley, R. M., Gersbach, C. A. and Barbas, C. F. 3rd. (2009). Synthesis of programmable integrases. Proceedings of the National Academy of Sciences of the United States of America. 106: 5053-5058.
    12. Grainge, I. and Jayaram, M. (1999). The integrase family of recombinase: Organization and function of the active site. Molecular Microbiology. 33(3): 449–456.
    13. Grindley,N.D.F., Whiteson,K.L. and Rice,P.A. (2006). Mechanisms of site-specific recombination. Annual Review of Biochemistry. 75: 567–605.
    14. Mercer, A. C., Gaj, T., Fuller, R. P. and Barbas, C. F. (2012). Chimeric TALE recombinases with programmable DNA sequence specificity. Nucleic Acids Research. 40: 11163–11172.
    15. Prorocic, M. M., Wenlong, D., Olorunniji, F. J., Akopian, A., Schloetel, J. G., Hannigan, A., McPherson, A. L. and Stark, W. M. (2011). Zinc-finger recombinase activities in vitro. Nucleic Acids Research. 39: 9316–9328.
    16. Sancar,A., Lindsey-Boltz,L.A., Unsal-Kacmaz,K. and Linn,S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual Review of Biochemistry. 73: 39–85.
    17. Sarkar, I., Hauber, I., Hauber, J. and Buchholz, F. (2007). HIV-1 proviral DNA excision using an evolved recombinase. Science. 316: 1912-1915.
    18. Sirk, S. J., Gaj, T., Jonsson, A., Mercer, A. C. and Barbas, C. F. (2014). Expanding the zinc-finger recombinase repertoire: Directed evolution and mutational analysis of serine recombinase specificity determinants. Nucleic Acids Research. 42: 4755–4766.
    19. Smith,M.C. and Thorpe,H.M. (2002). Diversity in the serine recombinases. Mol. Microbiol. 44: 299–307.
    20. Thyagarajan,B., Guimaraes,M.J., Groth,A.C. and Calos,M.P. (2000). Mammalian genomes contain active recombinase recognition sites. Gene. 244: 47–54. 
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