Team:MIT/Part Collection

• ABOUT US
  Team Attributions
• BACKGROUND
  Alternative Splicing CRISPR RNA Binding Proteins REST
• PROJECT
  Guides and ASOs mKate-FF4 2 Exon mKate HBG 3 Exon mKate HBG 3 Exon Dual Fluorescence
• LAB WORK
  Parts/Improved Parts Protocols Notebook Future Work
• MODELLING
• HUMAN PRACTICES
  Integrated HP Public Engagement Collaborations InterLab
• AWARDS
  HP Silver HP Gold Education & Public Engagement Model Parts Collection

Parts Collection

Currently, the iGEM parts registry does not have a set of parts that allows team to efficiently knockdown and process RNA sequences. We have created a new parts collection that enables teams to do not only targeted alternative splicing control, but also any other post-transcriptional control. By using a new RNA-targeting CRISPR protein called Cas13a (BBa_K2429006-BBa_K2429011), teams can do sequence-specific targeting of RNA. Teams can generate the guides RNAs for targeting RNA sequences of interest, and then can either clone the whole gRNA into the U6 backbone (BBa_K2429055) or just the targeting region into the U6: LacZ DR backbone (BBa_K2429180), the latter of which allows for easy visual selection of properly cloned guides. Additionally, teams can generate anti-sense oligonucleotides (ASOs) or other short DNA sequences, and then clone them into the U6: LacZ Full backbone (BBa_K2429179). These U6 promoter constructs allow for constitutive expression of short RNA sequences in mammalian cells.

We built several variations of the Cas13a protein in addition to the protein itself (BBa_K2429006):

• BBa_K2429007: dCas13a: a catalytically deactivated form of L. shahii Cas13a
• BBa_K2429008: Cas13a-DDX6: the CRISPR protein with the DDX6 DEAD-box sequence attached
• BBa_K2429009: dCas13a-DDX6: a catalytically deactivated form of L. shahii Cas13a with the DDX6 DEAD-box sequence attached
• BBa_K2429010: NL-Cas13a: features the CRISPR protein flanked by nuclear localization sequences.
• BBa_K2429011: NL-dCas13a: features a catalytically deactivated form of L. shahii Cas13a flanked by nuclear localization sequences.

These parts exist as MammoBlock entry vectors. By using an LR reaction, one may put a promoter of their choice up stream of the Cas13a protein variant, given that the promoter is in the correct plasmid for said reaction.

These Cas13a variants can potentially give the user more control over the level of post-transcriptional modification based on the protein used, assuming each the Cas13a proteins' has a distinguishable effect.

Additionally, the associated target sequences for the Cas13a variants can be inserted in the backbone made especially for creating such guides, known as U6: LacZ DR (BBa_K2429180), which features a U6 promoter (BBa_K2429055) upstream of the where the guide would be inserted.

This collection also features the U6: LacZ Full backbone(BBa_K2429179), which allows for the production of anti-sense oligonucleotides by inserting the target sequence in the backbone, just like with the guides. The production of ASOs can serve as a control for how well the Cas13a protein is affecting splicing, and helps make more precise conclusions about whether the presence of the protein or the guide RNA is contributing to alterations in splicing or protein knockdown.