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.