Team:Macquarie Australia/Singapore

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Singapore iGEM Team

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After reading that we were continuing on from last year’s project, the NTU-Singapore team approached us offering to help us knock in our very large plasmids (>10kbp). After further discussion we decided against this option as it would be too hard to coordinate in the little time we had left (September to October).

Coincidentally, we started a CRISPR-Cas9 approach this year as well. This led to the sharing of ideas and developed into other possible collaborative work. For our CRISPR-Cas9 project, we managed to show in vitro that our gRNA-Cas9 complex was able to target and cleave the PCR product of our target gene, hemH. This assay also provides a quick and easy test to their Cas9, which was their overall goal. Our collaboration involved testing their Cas9 in bacterial cells which would help to characterise their Cas9 functions when compared against the commercial Cas9 we acquired for our own experiments.

To facilitate this, NTU-Singapore did a purification tag switch (FLAG to 6xHis) so that we could purify their mutants of interest with high yields for characterisation using our in vitro assay design. In return, they attempted to help us generate a hemH knockout, and perform RT-PCR on our Magnesium chelatase plasmid containing 6 genes, for which we have functional assays to show that it works.

In summary what we did for them:

1. Sent the NTU-Singapore team our Mg-chelatase operon plasmids and primers designed
   for each gene for the RT-PCR
2. Purified and characterised their Cas9 mutants of interest proving their mutant enzyme functions

What they did for us:

1. Sent us their Cas9 mutant plasmids after cloning in a His-tag
2. Attempted to generate hemH Knockout
3. Performed RT-PCR of the genes in our Mg-chelatase operon

Fig 1. Digests of the Ferrochelatase PCR product target, by the two Cas9 mutants, and the wild type Cas9.

Shown above are the in vitro confirmations of the functional CRISPR/Cas9 from NTU-Singapore. The three Cas9 proteins that were studied in this experiment: wild type (Wt), 459 mutant (BBa_K2130002), 462 mutant (BBa_K2130001). All three were seen to cut our Ferrochelatase PCR product target, amplified in earlier weeks. This digestion was confirmed against the negative control without Cas9, and the commercial Cas9, as the Ferrochelatase was seen to be accurately cleaved into a 850bp fragment and a 150bp fragment, the lower band of less than 100bp is the excess gRNA duplex. From our efforts, we have proven that their mutant Cas9 is functional in vitro, providing first evidence for the NTU Singapore team that these 2 mutants are functional nucleases.