Difference between revisions of "Part:BBa K3168001"

Latest revision as of 15:19, 21 October 2019

dCas9-CP1041

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease which is part of the CRISPR-immune system of bacteria such as Streptococcus pyogenes. This endonuclease is used a lot in research to facilitate efficient genome engineering (Ran, 2013). Cas9 can target any sequence by simply specifying a 20 nucleotide targeting sequencing within the guide RNA. dCas9 is a ‘dead’ variant of Cas9 because it does not have its endonuclease activity. This means that dCas9 binds dsDNA but does not cut the DNA, because of mutations in the RuvC1 and HNH nuclease domains (Park, 2017). The basic part dCas9-CP1041 is a circularly permuted (CP) variant of dCas9. This means that the order of the amino acids has changed, which results in a protein with different connectivity, but an overall similar three-dimensional structure. The original N- and C-termini are connected by a 20 aa linker which results in new termini at different locations compared to the original orientation (Figure 1). The new N-terminus of this dCas9 is asparagine 1041. Furthermore, dCas9-CP1041 only contains one cysteine (S867C) at the HNH domain to enable dye incorporation via maleimide coupling (Sternberg, 2015). Finally, no stop codon is included, so fusion proteins can be made by combining parts.

Figure 1. Principle of circular permutation (Oakes, 2019).

Usage and Biology

Different fusion proteins can be made with dCas9 using these new termini. According to Oakes 2019, the circular permutation of Cas9 provides an advanced platform for RNA-guided genome modification and protection (Oakes, 2019). The TU_Eindhoven team 2019 used the new orientation of the C-terminus to develop a fusion protein of dCas9 and NanoLuc. The cysteine in dCas9-CP1041 is used to couple a Cy3 dye via maleimide coupling. The new orientation of NanoLuc and the Cy3 incorporation allows the detection of conformational changes when dCas9 binds to DNA (BBa_K3168007). When the fusion protein binds to DNA, bioluminescence resonance energy transfer (BRET) occurs between NanoLuc and the incorporated Cy3.

References

Oakes, B. L., Fellmann, C., Rishi, H., Taylor, K. L., Ren, S. M., Nadler, D. C., ... & Savage, D. F. (2019). CRISPR-Cas9 circular permutants as programmable scaffolds for genome modification. Cell, 176(1-2), 254-267.

Park, J. J., Dempewolf, E., Zhang, W., & Wang, Z. Y. (2017). RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis. PloS one, 12(6), e0179410.

Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature protocols, 8(11), 2281.

Sternberg, S. H., LaFrance, B., Kaplan, M., & Doudna, J. A. (2015). Conformational control of DNA target cleavage by CRISPR–Cas9. Nature, 527(7576), 110.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 526
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 ===dCas9-CP1041===
-Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease which is part of the CRISPR-immune system of bacteria such as Streptococcus pyogenes. This endonuclease is used a lot in research to facilitate efficient genome engineering (Ran, 2013). Cas9 can target any sequence by simply specifying a 20 nucleotide targeting sequencing within the guide RNA. dCas9 is a ‘dead’ variant of Cas9 because it does not have its endonuclease activity. This means that dCas9 binds dsDNA but does not cut the DNA, because of mutations in the RuvC1 and HNH nuclease domains (Park, 2017). This basic part is a circular permuted (CP) variant of dCas9. This means that the order of the amino acids has changed, which results in a protein with different connectivity, but an overall similar three-dimensional structure. The original N- and C-termini are connected by a 20 aa linker which results in new termini at different locations compared to the original orientation (Oakes, 2019). The new N-terminus of this dCas9 is asparagine 1041. Furthermore, dCas9-CP1041 only contains one cysteine (S867C) at the HNH domain to enable dye incorporation via maleimide coupling (Sternberg, 2015). Finally, no stop codon is included, so fusion proteins can be made by combining parts.
+Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease which is part of the CRISPR-immune system of bacteria such as Streptococcus pyogenes. This endonuclease is used a lot in research to facilitate efficient genome engineering (Ran, 2013). Cas9 can target any sequence by simply specifying a 20 nucleotide targeting sequencing within the guide RNA. dCas9 is a ‘dead’ variant of Cas9 because it does not have its endonuclease activity. This means that dCas9 binds dsDNA but does not cut the DNA, because of mutations in the RuvC1 and HNH nuclease domains (Park, 2017). The basic part dCas9-CP1041 is a circularly permuted (CP) variant of dCas9. This means that the order of the amino acids has changed, which results in a protein with different connectivity, but an overall similar three-dimensional structure. The original N- and C-termini are connected by a 20 aa linker which results in new termini at different locations compared to the original orientation (Figure 1). The new N-terminus of this dCas9 is asparagine 1041. Furthermore, dCas9-CP1041 only contains one cysteine (S867C) at the HNH domain to enable dye incorporation via maleimide coupling (Sternberg, 2015). Finally, no stop codon is included, so fusion proteins can be made by combining parts.
 [[File:T--TU_Eindhoven--CPprinciple.png|300px|]]