Part:BBa_K2148013

Cas9 gene

This is the coding DNA sequence (CDS) for the Streptococcus pyogenes Cas9 protein. There is a Gly-Ser fusion linker at the end of the sequence to allow for the possibility of forming fusion proteins for verification purposes.

Usage and Biology

The Cas9 protein is part of the bacteria's CRISPR/Cas9 immune defense mechanism to identify and destroy foreign DNA. By incorporating the foreign DNA into the bacteria's own DNA, it has a memory of any prior foreign DNA that the bacteria has encountered. The Cas9 endonuclease along with the tracrRNA and crRNA (CRISPR RNA- Clustered Regularly Interspaced Short Palindromic Repeats) would cleave the foreign DNA which forms a 20-nucleotide crRNA complementary sequence.

Development in creating/specifying our own the desired 'target' cutting site (sgRNA), would enable site specific modification. As C.reinhardtii chloroplast has bacterial origin, it is envisioned of using the CRISPR/Cas9 mechanism to accelerate the propagation of an inserted plasmid. In addition, Non-Homologous End Joining is absent in chloroplast which enables the use of this method in the choloroplast.

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 3379
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Alex Reis, Ph.D., Bitesize Bio, Breton Hornblower, Ph.D., Brett Robb, Ph.D. and George Tzertzinis, Ph.D., New England Biolabs, Inc. CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology. NEB expressions Issue I, 2014

John G Doench, Ella Hartenian, Daniel B Graham, Zuzana Tothova, Mudra Hegde, Ian Smith, Meagan Sullender, Benjamin L Ebert, Ramnik J Xavier & David E Root. Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nature Biotechnology 32, 1262–1267 (2014), doi:10.1038/nbt.3026