Ten-year iGEM: 2015

How did you and your team come up with our project?

I think, for each iGEM team, the generation and establishment of our topic are the most highlight moment. The first time I hear the idea of this project is when, in a sunny afternoon, Bohou Wu asked us whether we could use the CRISPR / Cas9 system to monitor the genes in the cells. This bold idea actually comes from an article published then, called "Programable RNA tracking in live cells with CRISPR / Cas9" (Nelles D et al., Cell., 2016). In this paper, Yeo Gene’s group design a CRISPR / Cas9 system that could report RNA localization in living cells. They take advantage of dCas9-GFP fusion protein without catalytic activity, sgRNA targeting intended RNA and PAM sequence (PAMmer) that cannot be easily degraded.

In order to use this system to monitor mRNA mutations, Bohou and Yiming Huang first proposed that we can fuse nuclear localization sequence (NLS) to dCas9. We introduced the sgRNA and PAMmer in the paper mentioned above, to allow dCas9 to recognize mRNA. This makes it limited in the cytosol, while it will show less affinity to the mRNA, thus enter the nucleus, when mRNA is mutated.

But how could we monitor mRNA mutation with dCas9’s viable affinity to it? Xinzhi Zou suggested that we could use a new screening marker, thymidine kinase. Different culture medium can force yeast, expressing thymidine kinase, to live or die, so it would be easy to accomplish both positive and reverse screen. To couple the dCas9-binding system with the screening system, we also fused dCas9 with transcription activator Gal4, and add Gal4-recognising sequence on the upstream of thymidine kinase coding sequence. Consequently, the localization of dCas9 determines it functions—in the cytosol, it monitors mRNA mutation; while in the nucleus, it turns on the suiciding system—and we can force the cell to convict suicide when mRNA mutates. The system is named suvCas9 (short for “Surveillance dCas9).

However, such a system has a problem in practical application—the special PAM sequence consists of a mixture of DNA and 2-methoxy modified RNA, which makes its synthesis requiring too much time and cost. This forced us to stop without any alternative plan, until our advisor Junbiao Dai provided a cool solution to ideas: let the cell to synthesize PAMmer by themselves. This idea comes from the literature "Genomically encoded analog memory with precise in vivo DNA writing in living cell populations" (Farzadfard F et al., Science, 2014), in which the system, containing a special reverse transcriptase, a primer sequence and a template sequence (containing the sequence of target single stranded DNA), meet the cell’s demand to synthesize ssDNA. Since the component of the system is in a single plasmid and it would be easy to change the template sequence, we are overwhelmed by the elegance and simplicity of the system and decided to use it. Here, a complete project was born.

How did you and your team come up with our project?

Determination of project in 2015 suffered a lot of twists and turns. We wondered straw power generation and heavy metal detection, mussel mucin and E. coli imaging. The final decision, Construction of CRISPR/Cas9 Based Real-time Digital Data Storage System in E.coli, came just before we went to the Jamboree. Yiming Huang, inspired by a Nature Method paper which use recombinase to store and edit information in E.coli genome (Yang L, Nielsen A A K, Fernandez-Rodriguez J, et al. Permanent genetic memory with> 1-byte capacity[J]. Nature methods, 2014, 11(12): 1261-1266.), thought we can use sgRNA and dCas9 to replace the DNA-recognizing domain of Cre, to get unlimited recombinase and store a large amount of information, theoretically. Other team members thought we can use light-sensitive switch to control recombinase. After a series of design and debates, our project was born. Surprisingly, a Nature paper published in 2017 reported their information storage and edition in E.coli (Shipman S L, Nivala J, Macklis J D, et al. CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria[J]. Nature, 2017, 547(7663): nature23017.), which also used CRISPR and has a similar core design with us. Such a coincidence makes us, far from that of PhD students, feel very happy.

Are there any regrets at that time?

There are of course a lot of regrets, including our failure in dCas9-recombinase function test, in blue- and green-light switch, in parts’ function testing, and also in enough barbeques and karaoke. But it’s indeed these regrets that impress us with days spent together and illuminate our junior successors.

What do you think about iGEM and synthetic biology, having participated in it for two years? Is there any change in your views?

iGEM is a great platform for us. For me, biological knowledge is applied to build systems and tools to solve the problem. However novel your idea is, you can test it in iGEM. Upon participating in iGEM, I not only learn more skills and meet more friends, but also believe more and more that once you want to do well in something, you need to spare no efforts in it.

What kind of team do you think Tsinghua iGem 2015 is?

Tsinghua iGEM 2015 team is, due to lack of experience and guidance, a little immature. Every time we were confronted with problems, we just didn’t know what to do. And we also got much criticism from teachers. However, this made us united and insisted with our love for synthetic biology and intrinsic persistence. After many sleepless nights, we finally got satisfying results and we are very happy.

What suggestions would you like to give to the junior successors of Tsinghua?

Synthetic biology is cool, iGEM is cool, and Tsinghua iGEM team is even more cool. Come here to practice your ideas, and you will feel rewarding!