Team:Moscow City/Contribution

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We made a contribution for future iGEM teams
Our contribution was based on the challenges our team faced in the beginning


We have developed tutorials to help understand UGENE, SNAPGENE, and DNA or RNA analyses. We've had our own difficulties with these programs for our purposes, and we decided to help iGEM beginners.

Download them:
Review of recent years
diagnostics projects on iGEM

When we started the project, we had a hard time choosing a diagnostic method: a unique, most appropriate way to diagnose the virus, related to synthetic biology. We also wanted it to be new for iGEM, so we looked at the previous iGEM projects and made the analytical table. It will help all new teams to get oriented.

We have characterized PspCas13b (BBa_K2807002).

We would like to thank the NUS Singapore-Sci (2018) iGEM team for presenting the design of their RNA base editor project based on the PspCas13b protein (BBa_K2807002). We have investigated several articles on this protein and we would like to add important features of PspCas13b.

A possible solution to off-target activity by engineering dCas13b (catalytically inactive nuclease) fused to an RNase domain (such as the PINc domain) to generate a dCas13–PIN platform capable of precise targeting and controlled RNA degradation [1].

It is also worth paying attention to spacers: data indicated that crRNA requires >21-nt base-pairing with its target to trigger the necessary CRISPR-Cas conformational change and nuclease activation necessary for target degradation and predicts low probability of off-target activity transcriptome-wide due to the extensive (>21-nt) spacer-target base-pairing required. Importantly, the ability of PspCas13b to tolerate up to 3-nt mismatches, especially in internal regions, indicates its potential to remain effective against the majority of variants with single-nucleotide polymorphisms in the target sequence, conferring protection against viruses such as SARS-CoV-2. [2]

Some data showed that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays [3]. Also, when the length of Cas13b gene needs to be reduced, e.g. to not exceed size limits of AAV's cargo capacity, HEPN2 gene can be removed - as it is appears to be significantly less integrated into the overall structure than the other domains, Cas13b enzyme with HEPN2 removed would retain RNA targeting activity for base editing [4].
1 - Ali et al. CRISPR/Cas13 as a Tool for RNA Interference. Trends Plant Sci. 2018 May;23(5):374-378.
2 - Fareh et al. Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance. Nat Commun 12, 4270 (2021).
3 - Mahas et al. CRISPR-Cas13d mediates robust RNA virus interference in plants. Genome Biol 20, 263 (2019).
4 - Slaymaker et al. High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage. Cell Rep. 2019 Mar 26;26(13):3741-3751.e5.