Knockout and Characterization of LC2 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 quantitative PCR (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. [2]

We can utilize this highly efficient system to characterize the role of LC2 in the movement of T. brucei by introducing a cut in the LC2 gene. If this component of axonemal dynein is no longer expressed, we can compare it to wild type movement to characterize its role.

The efficiency of this knockout can also be assessed using 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 [3]. This technique will allow for relative quantification of differential gene expression between wild-type and knockout Trypanosome cell lines.

Materials and Methods

gRNAs were made using a combination of the TriTryp database [4], the Eukaryotic Pathogen CRISPR Guide RNA Design Tool (EuPaGDT)[5], LeishGEdit [6], and Geneious software [7].

To design the knockout guide we inputed 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.

In order to assess the validity of the anticipated CRISPR/Cas9 knockout, a two-step RT-qPCR standard curve method was adopted for comparison  of mRNA expression between wild-type and LC2+FLAM3 RNAi double knockdown cells. Total RNA was extracted from raw cell lysate incubated with DNase I, and the efficacy of the primers was confirmed through a two-step workflow of cDNA synthesis through reverse transcription, followed by traditional PCR amplification. Real-time qPCR with a five-point standard curve was used to determine the efficiency of the reaction. The technique was then applied to a one-step RT-qPCR workflow to streamline the process and ultimately compare RNA expression between the two biological groups.

Results

When generating gRNA using EuPaGDT [6], the tool reported no potential off target effects and a high level of efficiency for the guides.

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.

One-step RT-qPCR of wild-type and FLAM3+LC2A double knockdown RNA resulted in consistent Cq values across replicates for a five-point standard curve from 1 x 10^2 to 1 x 10^-2 ng/μL. However, reaction efficiencies were exceptionally low, as indicated by the steepness of the curve. Furthermore, comparison of fold change in Cq values did not reveal significant differences in mRNA expression between wild-type and knockout cells for a knockout that has already been validated by other experimental means.

Future Directions

Protocols will continue to be optimized for a successful transfection. Additionally, current RT-qPCR methods will attempt to demonstrate differential expression between wild-type and LC2+FLAM3 RNAi double knockdown cells. Soon thereafter, these methods will be modified with the goal of developing a successful qPCR assay for future use in CRISPR knockouts.

References

  1. Liu, B., Saber, A., & Haisma, H. J. (2019). CRISPR/Cas9: A powerful tool for identification of new targets for cancer treatment. Drug Discovery Today,24(4), 955-970. doi:10.1016/j.drudis.2019.02.011
  2. Rico, E., Jeacock, L., Kovářová, J., & Horn, D. (2018). Inducible high-efficiency CRISPR-Cas9-targeted gene editing and precision base editing in African trypanosomes. Scientific Reports,8(1). doi:10.1038/s41598-018-26303-w
  3. Real-Time PCR Handbook. Applied Biosystems. Thermo Fischer Scientific Inc., 2014.
  4. TriTrypDB. March 2, 2020. NIAID Bioinformatics Resource Centers. https://tritrypdb.org/tritrypdb/app
  5. Duo Peng and Rick Tarleton. EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens. Microbial Genomics. 2015. doi: 10.1099/mgen.0.000033
  6. Beneke T., Madden R., Valli J., Makin L., Sunter J. and Gluenz E (2017). A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. Royal Society Open Science 10.1098/rsos.170095.
  7. Geneious Prime 2020.1. https://www.geneious.com

Acknowledgements

-Research reported in this poster was supported by NIAID of the National Institutes of Health under award number R15AI137979.
-The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
-Research reported in this poster was supported by Clemson University through the Creative Inquiry program.