CyanoCapture and the McCormick lab have co-developed CRISPR-Cas tools for high throughput screening and selection of novel genetic factors involved in CO2 fixation, and further applied targeted engineering biology and metabolic engineering to develop cyanobacteria strains to suit industrial deployment. The initial phase included the adoption of existing cyanobacterial CRISPR interference (CRISPRi) systems to our fast strain and the development of this strain as a molecular chassis for the stable expression of desirable proteins. This was followed by the identification of novel genes in the carbon fixation pathway using high throughput CRISPRi screening and selection systems. The next phase included targeted marker-free gene engineering (deletion or overexpression) of desirable CO2 fixation pathway or regulatory genes, followed by genotype and phenotype characterisations. The final step of the project included the optimisation of protein expression and the growth of the engineered strains and demonstration of the enhanced CO2 fixation.

 In most published studies, carbon fixation was increased by modulating the expression of one or several genes (Daneshvar et al. 2022). Here, we considered higher-throughput methods to expand the scope of genetic engineering, for example, to target genes that were not fully annotated. Our second innovation was applying engineering biology techniques to maximise internal CO2 fixation combined with antibiotic marker deletion for outdoor deployment. This has led to IP on the Methods for markerless cloning using cre-lox.

Recent advances in CRISPRi tools provided an opportunity for high throughput genetic screening (Yao et al. 2020). With CRISPRi, genes were specifically targeted for repression within a genome. The principle was based on the CRISPRi/dCas9 system in which an inactive Cas endonuclease was guided to a specific sequence by a single guide RNA (sgRNA), which blocked gene expression. Our approach was highly innovative -- building on an inducible CRISPRi system from the McCormick lab, we synthesised sgRNAs targeting thousands of putative genes in our strain and assembled a pooled library of CRISPRi mutants. By measuring growth and CO2 fixation in the induced CRISPRi mutant population, and sequencing the DNA pool of the population, it is now possible to identify important genes that had not been identified through conventional forward genetics. Once determined, these present opportunities to build a combination of modified strains through marker-free genetic engineering, providing novel cell-factories that could be used commercially for CO2 capture or biomanufacturing.