Difference between revisions of "Part:BBa K2361001:Design"

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===Design Notes===
 
===Design Notes===
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After obtaining the dCas9 in a biobrick compatible form, this was used as a backbone to create dCas9 VRER. Multiple mutations were made in the C-terminal end of the proteins sequence. These mutations were implemented by exchanging the C-terminal portion (1590bps)
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This part originates from <i>Streptococcus pyogenes</i> Cas9 and it contains 7 mutations. Two amino acid mutations to catalitically inactivate it ( D10->A & H840->A) were already present in the part we started with.
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To remove the EcorI site an A->T substitution was made at position 1291, this mutation preserves the Ile codon. To change the PAM preference a set of four amino acid substitutions was performed (D1135V, G1218R, R1335E, T1337R).
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Besides the above mentioned mutations the spCas9 sequence was preserved.
  
 
===Source===
 
===Source===

Revision as of 16:42, 26 October 2017


dCas9 VRER


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1100
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3379
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part originates from Streptococcus pyogenes Cas9 and it contains 7 mutations. Two amino acid mutations to catalitically inactivate it ( D10->A & H840->A) were already present in the part we started with. To remove the EcorI site an A->T substitution was made at position 1291, this mutation preserves the Ile codon. To change the PAM preference a set of four amino acid substitutions was performed (D1135V, G1218R, R1335E, T1337R). Besides the above mentioned mutations the spCas9 sequence was preserved.

Source

This part originates from the plasmid pJWV102-PL-dCas9, which is available via Addgene (https://www.addgene.org/85588/). this part was made biobrick compatible by removing the EcoRI-site and adding the prefix. Finally the suffix and the VRER mutations were added by replacing the last part of dCas9 with a piece of synthetic DNA. The VRER mutations were described for hCas9 by Heler et al.

Heler, R. et al. Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response. Mol. Cell 65, 168–175 (2017)

References