CPF1-BASED CRISPR GENOME EDITING IN THE CYANOBACTERIUM N. PUNCTIFORME

CRISPR systems have been growing in their utility and their application throughout the biological field as researchers continue to grow in their understanding of the relatively novel genome editing technology. However, despite the potential of CRISPR as a genome editing tool, the complexity of applying this technology to a specific organism calls for custom modifications to the system to improve its success rate. In this project, a CRISPR-Cpf1 system that can be effectively employed in the cyanobacterium Nostoc punctiforme was designed, with a focus on the hormogonium development of this species. Multiple plasmids containing the CRISPR system and targeting different genes were constructed using a Gibson-based rapid assembly cloning method, and then were tested by introduction into Nostoc punctiforme via conjugation. Plasmids were constructed to mutate 7 different genes in N. punctiforme with 4 of the 7 successfully mutating their target genes. For one of the genes where the plasmid failed to produce mutants, the usage of a larger homology repair template (HRT) was found to enhance the efficiency of gene editing, allowing the gene to be knocked out. Thus, the length of the HRT appears to be a critical factor in designing successful constructs. The system developed in this project aims to make CRISPR a more viable tool in studying Nostoc cyanobacteria, and more specifically to aid in understanding the mechanisms behind hormogonium development in the studied species. This system may have a wider application for studying the Nostoc genus and related organisms, such as Anabaena