Difference between revisions of "Part:BBa K3776019"
 
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<partinfo>BBa_K3776019 short</partinfo>
 
<partinfo>BBa_K3776019 short</partinfo>
  
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Designed by Vo et al. (2020), this protein complex includes transposases TnsA, TnsB, TnsC and Cas enzymes Cas 8, Cas7, Cas6. These proteins are guided by a crRNA prefix flanked by two repeats. Users should clone their crRNA between these two repeats. This RNA guided protein complex inserts a donor DNA located between two flanks behind the into the genome of the host organism. Users should clone their genes of interest between these two flanks.  
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The INTEGRATE system stands for the insertion of transposable elements by guide RNA–assisted targeting system. Designed by Vo et al. (2020), this system includes a crRNA which guides the TniQ-Cascade and tnsA-tnsB-tnsC operon to insert a donor DNA into a site in an organism's genome. A single promoter drives the expression of the entire system, and can be switched by users with restriction enzymes. The 32bp crRNA is seamlessly flanked by two repeats and users should use BsaI restriction enzymes to change the crRNA. An online tool is provided by the publisher for users to generate suitable crRNA for use in the INTEGRATE system, please refer to the code availability of the article published by Vo et al. (2020). The TniQ-Cascade complex consists of the proteins TniQ, Cas8, Cas7 and Cas6, which together is guided by the crRNA to search the cell for matching DNA sites and to bind the DNA. After the TniQ-Cascade binds to the DNA and forms an R-loop, TniQ recruits the protein TnsC. Meanwhile, TnsA and TnsB binds and brings together the left and rights ends of the donor DNA and then are recruited by TnsC. The donor DNA is flanked by the 111bp Right end and 145bp Left end, users should clone their genes of interest between these two flanks. The donor DNA is finally inserted around 50bp downstream of the crRNA target sequence in the genomic DNA of the organism.(Vo et al., 2020)(Klompe et al., 2019)
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===References===
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Vo, P. L. H., Ronda, C., Klompe, S. E., Chen, E. E., Acree, C., Wang, H. H., & Sternberg, S. H. (2020). CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nature Biotechnology, 39(4), 480–489. https://doi.org/10.1038/s41587-020-00745-y
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Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S., & Sternberg, S. H. (2019). Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature, 571(7764), 219–225. https://doi.org/10.1038/s41586-019-1323-z
  
 
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Latest revision as of 09:27, 18 October 2021


INTEGRATE system

The INTEGRATE system stands for the insertion of transposable elements by guide RNA–assisted targeting system. Designed by Vo et al. (2020), this system includes a crRNA which guides the TniQ-Cascade and tnsA-tnsB-tnsC operon to insert a donor DNA into a site in an organism's genome. A single promoter drives the expression of the entire system, and can be switched by users with restriction enzymes. The 32bp crRNA is seamlessly flanked by two repeats and users should use BsaI restriction enzymes to change the crRNA. An online tool is provided by the publisher for users to generate suitable crRNA for use in the INTEGRATE system, please refer to the code availability of the article published by Vo et al. (2020). The TniQ-Cascade complex consists of the proteins TniQ, Cas8, Cas7 and Cas6, which together is guided by the crRNA to search the cell for matching DNA sites and to bind the DNA. After the TniQ-Cascade binds to the DNA and forms an R-loop, TniQ recruits the protein TnsC. Meanwhile, TnsA and TnsB binds and brings together the left and rights ends of the donor DNA and then are recruited by TnsC. The donor DNA is flanked by the 111bp Right end and 145bp Left end, users should clone their genes of interest between these two flanks. The donor DNA is finally inserted around 50bp downstream of the crRNA target sequence in the genomic DNA of the organism.(Vo et al., 2020)(Klompe et al., 2019)

References

Vo, P. L. H., Ronda, C., Klompe, S. E., Chen, E. E., Acree, C., Wang, H. H., & Sternberg, S. H. (2020). CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nature Biotechnology, 39(4), 480–489. https://doi.org/10.1038/s41587-020-00745-y

Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S., & Sternberg, S. H. (2019). Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature, 571(7764), 219–225. https://doi.org/10.1038/s41586-019-1323-z

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 1578
    Illegal NheI site found at 3665
    Illegal NheI site found at 7783
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1004
    Illegal BglII site found at 1795
    Illegal BglII site found at 2325
    Illegal BglII site found at 7570
    Illegal BamHI site found at 147
    Illegal BamHI site found at 8698
    Illegal XhoI site found at 8681
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 7702
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 113
    Illegal BsaI.rc site found at 87
    Illegal SapI.rc site found at 3081