Comparing different delivery systems in site-specific genome editing in Chlorella vulgaris

Abstract

Microalgae, such as Chlorella vulgaris, are among the most important eukaryotic microorganisms due to their ecological, biotechnological, and sustainable energy applications. Although the genome of C. vulgaris has been sequenced, its full genetic potential remains underutilized due to challenges in efficient genome editing. In this research, we aim to optimize the CRISPR-Cas9 genome editing system to achieve site-specific knockouts in the C. vulgaris genome. Two different delivery methods for CRISPR components plasmid-mediated transformation and direct ribonucleoprotein delivery were compared in terms of efficiency. Three guide RNAs targeting the nitrate reductase gene were designed and tested. The objectives were to assess mutation efficiency, validate phenotypic changes, and develop a genetic toolbox for C. vulgaris, enhancing its potential for applications in biotechnology. Phenotypic analysis confirmed successful gene disruption in transformants, while sequencing results highlighted the challenges in genotypic confirmation, emphasizing the need for improved protocols. This study contributes to advancing genetic engineering tools for C. vulgaris, providing a foundation for future biotechnological applications.