Knockout and Characterization of LC2 Knockdown in Axonemal Dynein of Trypanosoma brucei Using CRISPR-Cas9 and RT-qPCR

Ethan Lopez (1), Madison Ragland (2), Katherine Wentworth (2,4), Lucy Fischer (1), Subash Godar (3,4), Joshua Alper (2,3,4)

(1)Department of Genetics and Biochemistry, (2)Department of Biological Sciences, (3) Department of Physics and Astronomy, (4) Eukaryotic Pathogen Innovation Center

Abstract

Flagellar motility is a critical component of kinetoplastid pathogenicity, and it exhibits a unique tip-to-base wave propagation dependent on axonemal dynein motor proteins. Our recent, preliminary results suggest that axonemal dynein light chain 2 (LC2) regulates flagellar motility. However, LC2’s specific role in flagellar motility has not been well characterized. Therefore, we designed single-guide RNAs (sgRNAs) with no predicted off-target effects using the Eukaryotic Pathogen CRISPR Guide RNA Design Tool to knockout LC2 homologs in T. brucei using the CRISPR-Cas9 system. Additionally, we designed a sgRNA to tag the C-terminus of LC2 with GFP, His6, and BCCP for conjugation in in vitro single-molecule biophysical experiments. We ligated this guide RNA the pT7 vector, transformed into E. coli, and amplified through maxiprep. Currently, we are transfecting the vector into T. brucei. Furthermore, we are using reverse transcriptase quantitative PCR (RT-qPCR) to compare relative gene expression between wildtype, knockout, and previously developed RNAi knockdown cell lines for validation. The knockouts will allow us to characterize the role of LC2 in the motility of T. brucei cells. Ultimately, we expect our understanding trypanosome cell motility will provide a platform from which novel therapeutics can be developed.

Introduction

The bacterium S. pyrogenes developed the CRISPR-Cas9 system to protect against bacteriophages by carrying short segments of the phage DNA in its chromosomes as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). These segments become the part of the gRNA that interacts with tracrRNA to recruit the Cas9 endonuclease and help it find where in the genome to cut.

We can utilize this system to characterize the role of LC2 in the movement of T. brucei by introducing a cut in the LC2 gene.

gRNA was designed to disrupt the LC2 gene. The pT7 vector will be used to help deliver the gRNA into T. brucei through transfection.

Materials and Methods

gRNAs were made using a combination of the TriTryp database, the Eukaryotic Pathogen CRISPR Guide RNA Design Tool (EuPaGDT), LeishGEdit, and Geneious software.

To design the knockout guide I simply input the genomic sequences from TriTryp database into EuPaGDT. These results were then put into the pT7 vector using Geneious. This guide leads Cas9 to the middle of the LC2 gene.

These inserts were then hybridized, ligated into the pT7 vector, and transformed into E. coli cells. The E.Z.N.A Mini Kit II was used to mini prep the colonies from transformation. This product was then digested in order to remove the inserted sequence from the pT7 plasmid for sequencing analysis at the Bbs1 cut sites. To prep the plasmid for transfection, maxi prep was done in order to isolate the plasmid DNA.

Results

When generating gRNA using EuPaGDT, the tool reported no potential off target effects and a high level of efficiency for the guides.

After the Restriction Digest, constructs were submitted for sequencing.

Sequence results confirmed that restriction digest was successful, therefore, maxi prep was performed on the samples. Concentrations of the vector after this procedure were found to be approximately 623.4 ng/μL for the alpha homolog and 1021.4 ng/μL for the beta homolog.

Future Directions

Transfection protocols will continue to be modified until a successful one is made. Once this is completed, the knockout efficiency will be tested using RT-qPCR. Quantitative PCR (qPCR) is used to quantify gene expression through measuring the amount of DNA produced in a real-time PCR reaction by way of a fluorescent DNA-binding dye [4]. This technique will allow us to compare relative quantification of differential gene expression between wild-type and knockout Trypanosome cell lines. Currently, our efforts have been focused on using one-step reverse transcriptase PCR (RT-qPCR) to compare mRNA expression of wild-type and LC2+FLAM3 RNAi double knockdown cells with the goal of developing a successful assay for future use in CRISPR knockouts.

References

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  3. Real-Time PCR Handbook. Applied Biosystems. Thermo Fischer Scientific Inc., 2014.
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