Team:IISER Berhampur/Contribution
New part added to the CRISPR Collection in Parts Registry!
In recent years, CRISPR/Cas technology has gained momentum with its wide-ranging applications in synthetic biology. It evolved from the wild-type spCas9 having natural guide RNAs with inefficient cleavage mechanisms to different types of Cas9, Cas12, and Cas13 proteins with improvised endonuclease activity. But sometimes, scientists face problems such as the limited applicability of this technique due to Protospacer Adjacent Motif (PAM) dependent mechanism or it can be the size of the Cas protein which makes it difficult to deliver them in cellular systems. Also, some of these nucleases can have off-target effects. To tackle these problems, we wanted to use a highly efficient and robust technique in our project CODE M.
While on our journey to bring up project CODE M, we explored various endonucleases and decided to use the Cas14a1 nuclease, a recently discovered endonuclease of the CRISPR/Cas family. It has an exceptionally compact size with just 400-700 amino acids instead of typical >1000 amino acids. It can cleave ssDNA without any PAM specificity which provides flexibility for its use in the lab. Also, target recognition by Cas14 triggers non-specific cutting of ssDNA molecules, an activity that enables high-fidelity SNP genotyping. Also, the engineered sgRNAs used with this protein are smaller.
Though we couldn’t fully complete the lab work due to lockdown restrictions, we have provided the complete design information and the necessary protocols to use the recombinant protein. Part BBa_K3982001 has been added to the Parts Registry with its documentation. This will be a useful contribution to the future iGEM teams who intend to use CRISPR/Cas14 technique in their projects.
Here are a few other parts where we have used Cas14a1.
| Description of the construct | Part Number | Links |
|---|---|---|
| 6xHis - Tagged Cas14a1 with TEV site | BBa_K3982006 | http://parts.igem.org/Part:BBa_K3982006 |
| CODE M Construct C1 | BBa_K3982025 | http://parts.igem.org/Part:BBa_K3982025 |
| CODE M (Standard) Construct S1 | BBa_K3982026 | http://parts.igem.org/Part:BBa_K3982026 |
Further, there are many other relevant parts for Cas14a1 which can be found on the Parts https://2021.igem.org/Team:IISER_Berhampur/Parts page.
New documentation for BBa_K2323010
While we were conceptualizing our project idea, our team went through countless literature surveys and finally decided to use CRISPR/Cas14a in our project due to its miniature size and high-fidelity single nucleotide polymorphism genotyping. During literature surveys, we also explored the potential of Cas13a as a tool for SNP genotyping. Later, we came across its excellent property as a tool for RNA targeting.
Team iGEM Munich 2017 had used LwaCas13a, an ortholog of Cas13a for cleaving RNA in-vitro to combat antibiotic resistance in their project “CascAID.” We learned from the literature that LwaCas13a protein fused with msfGFP can be engineered for in vivo use in human HEK293FT cells to target transcripts in live cells of both plant and animal origin. Also, we explored how other orthologs of Cas13a can be used to impart different targeting efficiencies.
Phylogentic analysis for MDR-TB
While analyzing MDR-TB mutation patterns we found that while phylogenetic data for TB was abundant there is a very limited amount of data on phylogenetic analysis for MDR-TB. So we decided to produce our own phylogenetic data and visualize it via a phylogenetic tree. The phylogenetic tree can be used by other iGEM teams and people interested in analysing the evolution of strains having MDR-TB mutations.
Phylogenetic trees -
Troubleshooting
Troubleshooting for Phylogenetic analysis -
Alignment tools
We constructed alignments using multiple software including the K-align and MAFFT. We also tested multiple alignment strategies (G-INS-I, Q-INS-I, L-INS-I, and E-INS-I) as implemented in the alignment software. We had initially used the K-align software however, a segmentation error (core dump) occurred since our system did not have enough memory to store the K-align files. The L-INS-I option as implemented in the MAFFT created a relatively better alignment with fewer gaps and this was further improved by correcting the alignment using TextPad.
Maximum Likelihood trees
NCBI BLAST Search
We first filtered our protein blast results only for Mycobacterium tuberculosis (tax id 1773) but the sequences obtained were redundant and many of them were fragmented. We then limited our searches to the Mycobacterium taxa (tax-id 1763). As such the searches recovered sequences across all Mycobacterium taxa and only the sequences belonging to Mycobacterium tuberculosis and Paratuberculosis strains were considered for further analysis. This gave us a much more non-redundant and less fragmented set of sequences.
