Applying CRISPR for Genome Editing and Increasing Flux Towards Products in Cyanobacteria

CRISPR is a powerful tool for sequence specific selection and controlling gene expression, but implementation is not always straight forward even in bacteria. Using CRISPR paired with recombineering has become routine for gene knockouts, knock-ins, and precise genomic changes in Escherichia coli. We attempted to use CRISPR in industrially relevant fast growing cyanobacteria Synechococcus sp. strain PCC 7002, which could be used to turn carbon dioxide and sunlight into valuable products. We optimized a CRISPR interference system for tunable gene repression in cyanobacteria and were able to increase flux towards a desired product. By repression of a native nitrogen assimilation gene we were able to increase lactate production by 2-fold compared to a base engineered strain. Attempts for using CRISPR for genome editing in cyanobacteria has proved to be challenging. It has been reported that Cas9 is toxic to cyanobacteria, but we have made strains that express active Cas9. Initial results show that Cas9 seems to be cleaving the intended target, but we are unable to get the intended genomic edit. There seem to be specific challenges for using CRISPR for genome editing in some organisms including toxicity, reliance on specific DNA uptake mechanisms, lack of appropriate expression systems, and coordinated expression of both Cas9, guide RNA, and repair template.