Design

The process of storing information in DNA involves consideration of the environment in which the DNA generated is stored. Key traits of an appropriate storage medium include the ability to preserve the existing information, additional post-processing costs and steps, and physical space requirements. [1]. In vivo DNA storage is beneficial to copy and convenient to use. In addition, in vivo information editors (Crispr/Cas9, etc.) are more conducive to regulation and go into effect.. In our project, DNA in vivo storage is undoubtedly more conducive to the role of base editor. Considering the compatibility of the whitelist given by iGEM with the CRISPR system, we selected Saccharomyces cerevisiae as the engineering strains for information storage and multi-site editing.

To achieve multi-site editing efficiency, it is necessary to synthesize an ultra-long gRNA array and transfer it into Saccharomyces cerevisiae cells. It is difficult to synthesize and assemble ultra long gRNA array because of its highly repetitive sequence. In addition, releasing sgRNAs from polycistron is also a challenge. Based on Nicholas S. McCarty's review[2], we used tRNA processing mechanism to release gRNAs from array. The gRNA arrays flanked by tRNAs can be transcribed and processed by endogenous RNase P and RNase Z, which cut the 5' and 3' ends respectively, of pre-tRNAs, to produce functional gRNAs. At the same time, each polycistron consists of 3 gRNAs, 4 tRNAs, synthetic promoters and terminators.

In one transcription unit,we used synthetic unrepeated type II promoter to express gRNAs and each gRNA is flanked by different kinds of tRNA. In this way,we decreased repetition level of our gRNA array.

We finally constructed a 30-gRNA array which contains 10 transcription units. Theoretically, this gRNA array combined with CBE can achieve simultaneous editing of 30 sites.

For more accurate editing, based on the advisors' project, we chose the high-precision base editor —— nCDA1Δ198-BE3, which was developed by Junjie Tan et al. This base editor has a narrower editing window and more accurate editing capabilities, which is very suitable for our project.

In order to verify the editing ability of our 30-gRNA array, we designed 30 guides targeting 5 genes of violacein pathway that we have already integrated into the yeast genome.。This mutation will prevent yeast from producing violacein.

Sequencing can be used to characterize editing results, but sequencing all colonies is time-consuming and laborious. Our team designed three gRNAs targeting ade2 on chromosome XV of Saccharomyces cerevisiae as a marker to show whether editing has occurred. Editing on ade2 will make it lose function, and such colony will appear red on the SC -ade media[4]. We randomly selected some red colonies to further validate the editing results via Sanger sequencing.

After receiving the sequencing results, it will be aligned with the designed sequence. The number of editing events on each site and the number of editing sites for a single colony will be used to evaluate the editing ability of our editing system. At the same time, the sequencing results can be inputted into the decoding software, which will verify whether expected post editing information is obtained.