Characterization of LC2 in Axonemal Dynein of Trypanosoma brucei Using CRISPR-Cas9 Modifications

Madison Ragland (1), Kathrine Wentworth (1), Subash Godar (2,3), Joshua Alper (1,2,3)

(1) Department of Biological Sciences, (2) Department of Physics and Astronomy, (3)Eukaryotic Pathogens Innovation Center

Abstract

Kinetoplastids are a class of flagellated eukaryotic protists, including Trypanosoma and Leishmania, that threaten over 350 million people globally. Axonemal dynein is critical to the flagellar beat of T. brucei and has a central subunit, LC2, that we target. The role of LC2 in the function of dynein, and therefore in flagellar movement, has not been well characterized. Therefore, we designed guide RNAs (gRNAs) to program the CRISPR-Cas9 system to knock-out and tag LC2 homologs in T. brucei. gRNA leads the Cas9 endonuclease to different loci on the LC2 gene depending on the approach we wish to employ. To knock out the LC2 gene, we designed gRNA that targets and creates insertions and deletions in the middle of the gene. To tag LC2 with GFP, His6, and BCCP, we designed a gRNA that targets the end of the gene close to the 3’UTR using the Eukaryotic Pathogen CRISPR Guide RNA Design Tool, which reported no potential off target effects for the gRNA. Tagging will allow us to visualize LC2 in vitro and purify the axonemal dynein from trypanosome cells. The knock-out of the LC2 gene, with the efficiency of CRISPR-Cas9, will allow us to clearly quantify the role of LC2 in axonemal dynein and its impact on motility of T. brucei.The atypical flagellar beat observed in T. brucei can be better understood by applying CRISPR-Cas9 technology to the LC2 gene.

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.

I used a variety of methods, described below, to design gRNA for Knockout and Knock-in approaches. The plasmid design serves as a template for Homology Directed Repair (HDR) to knock-in GFP, His6, and BCCP.

CRISPR full res diagram

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.

Knockout:

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.

Knock-in:

A knock-in requires gRNA to lead Cas9 to the 3′ end of the gene and an HDR template to introduce the BCCP tag for column purification, His6 is for chromatography, and GFP for viewing with microscopy. Homology arms are introduced around these and are 220 base pairs in length to help with HDR efficiency. pT7 was again used as the vector backbone. gRNA flanks the homology arms in order to excise the HDR template, making it a double stranded insert. General knock-in plasmid format is shown below.

Results

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

Knockout gRNA for alpha and beta homologs were put into the pT7 vector shown below.

The knock-in gRNA flanks the outsides of the homology arms in the pT7 vector. GFP, His6, and BCCP tags were inserted between the homology arms. Collectively the insert was positioned approximately 200 bases away from the Origin of Replication. The HDR template was assembled using Geneious software and is shown below.

Once the gRNAs have recruited Cas9 we expect to see arrhythmic movement in the knockout cell line and small levels of green florescence in the knock-in cell line.

The following diagram shows the workflow for getting our constructs into T. brucei:

Screen Shot 2020-08-16 at 12.57.04 PM

Future Directions

After further experimentation, I will have data that helps us characterize the role of LC2 in the movement of T. brucei and shows how effectively the technology has been applied. In the future, we hope to see the gRNAs designed interact with Cas9 to knockout the LC2 gene and introduce the repair template containing GFP. A successful knock-in will enable localization of LC2, in vitro, via microscopy. The knockout will allow us to compare movement between wild type cells and cells with minimal amounts of LC2.

References

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