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 can be used either with the tracrRNA and crRNA (CRISPR RNA- Clustered Regularly Interspaced Short Palindromic Repeats) or with a guide RNA to cleave foreign DNA at a species locations. This makes exceptionally useful for DNA cloning in synthetic biology.

Use in Chlamydomonas reinhardtii chloroplasts

Cambridge-JIC 2016 team has developed a strategy that should, in principle, accelerate the process of achieving chloroplast homoplasmy -- potentially the most time-consuming step in chloroplast engineering. It depends on Cas9 selectively introducing cuts in untransformed chloroplast genomes to promote the spread of the the cassette of interest by homologous recombination (HR). This is possible, because Non-Homologous End Joining (NHEJ) is absent in chloroplasts, necessitating HR as the mechanism of repair. For more information, see the Cambridge-JIC 2016 Homoplasmy strategy.

Modifications to the original part

This part is based on the original Cas9 part in the registry, BBa_K1218011. To enhance the expression of Cas9 in the chloroplast chassis, we have codon-optimised it using the codon-optimisation softwaredeveloped by Saul Purton (UCL). We have also introduced 4 silent mutations to remove Phytobrick illegal restriction sites, recognised by Bsa1 and BsmB1 (see sequence annotation for the details of the mutations.

As our strategy involves the loss of Cas9 once homoplasmy is confirmed, Cas9 can be fused with the Verde FP (BBa_K2148007), so its absence can be confirmed by the loss of fluorescence. VFP is "coded" as a C-TAG, according to the Phytobrick standard – we have ensured that the fusion would be in the same reading frame. Finally, we have also added a C-terminal HA-tag so that the fusion can be analysed with Western Blots and other methods.

CAUTION: Previous attempts to express Cas9 in the nucleus Chlamydomonas reinhardtii have failed for unknown reasons.

Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii

We believe that the attempts to express Cas9 in the chloroplast of C. reinhardtii should be successful, as its environment is much closer to that in Streptococcus progenies , in which Cas9 originates.

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]

Compatiblity

Our Cas9 is coded as a CDS, according to the Phytobrick standard, and has to be fused to a promoter/5UTR/NTAG at 5', and a CTAG at 3'. We have developed a complementary Verde FP part, coded as a CTAG, to future teams to create Cas9-FP fusions quickly and efficiently.

We also submit psaA promoter/5UTR in our library (BBa_K2148004 -- with a flanking homology region, BBa_K2148000 -- without a homology region). It is a strong promoter that can be used to obtain good Cas9 expression yields.

Characterisation

We have confirmed that the insertion of Cas9 in L0 was successful by a restriction digest with Bsa1.

Verification by restriction digest, giving the expected bands

The ladders used above is Bioline 1kb and 100bp.

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

Jiang W, Brueggeman AJ, Horken KM, Plucinak TM, Weeks DP. Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii. Eukaryotic Cell. 2014;13(11):1465-1469. doi:10.1128/EC.00213-14.