Difference between revisions of "Part:BBa K3168000"

 
 
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__NOTOC__
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===dCas9===
  
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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). Furthermore, this part does not include a stop codon, so fusion proteins can be made by combining parts.
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===Usage and Biology===
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dCas9 can bind to target sequences without cutting the DNA. Therefore, dCas9 can be used to recognize specific genes or unique DNA sequences. A fusion protein of dCas9 with a luciferase and/or fluorophore can be used to visualize the binding of dCas9 to dsDNA.
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===Characterization===
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<strong>Expression</strong>
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<br>
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This part is optimized for expression of dCas9 in bacterial cells. The protein was successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC). All purification steps (lysate flow through, bind flow-through, wash flow-through, elution) and a concentrated sample of the elution were put on an SDS-PAGE gel to evaluate the purification process (Figure 1). dCas9 has a molecular weight of 158 kDa and a clear band is visible around 158 kDa in the elute. It is remarkable that there is also a band at this height in the wash flow-through. This indicates that the 30 mM imidazole of the wash buffer already causes the protein to be released from the column. Furthermore, a very prominent band around 158 kDa is visible in the sample of the concentrated elution, so dCas9 was successfully expressed. There are other bands visible, which means that the purity is not optimal. However, the contrast between the huge blobs and the other bands is big. 
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[[File:T--TU_Eindhoven--His-purificationd-Cas9.png|200px|]]
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''Figure 1. SDS-PAGE gel of Nickle affinity chromatography samples.''
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===References===
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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.
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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.
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===Sequence and Features===
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<partinfo>BBa_K3168002 SequenceAndFeatures</partinfo>

Latest revision as of 15:17, 21 October 2019

dCas9

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). Furthermore, this part does not include a stop codon, so fusion proteins can be made by combining parts.

Usage and Biology

dCas9 can bind to target sequences without cutting the DNA. Therefore, dCas9 can be used to recognize specific genes or unique DNA sequences. A fusion protein of dCas9 with a luciferase and/or fluorophore can be used to visualize the binding of dCas9 to dsDNA.

Characterization

Expression
This part is optimized for expression of dCas9 in bacterial cells. The protein was successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC). All purification steps (lysate flow through, bind flow-through, wash flow-through, elution) and a concentrated sample of the elution were put on an SDS-PAGE gel to evaluate the purification process (Figure 1). dCas9 has a molecular weight of 158 kDa and a clear band is visible around 158 kDa in the elute. It is remarkable that there is also a band at this height in the wash flow-through. This indicates that the 30 mM imidazole of the wash buffer already causes the protein to be released from the column. Furthermore, a very prominent band around 158 kDa is visible in the sample of the concentrated elution, so dCas9 was successfully expressed. There are other bands visible, which means that the purity is not optimal. However, the contrast between the huge blobs and the other bands is big.

T--TU Eindhoven--His-purificationd-Cas9.png

Figure 1. SDS-PAGE gel of Nickle affinity chromatography samples.

References

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.

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]