Design of an Effective sgRNA for CRISPR/Cas9 Knock-Ins and Full Mutant Segregation in Polyploid Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 is a highly promising organism for the production of diverse recombinant compounds, including biofuels. However, conventional genetic engineering in Synechocystis presents challenges due to its highly polyploid genome, which not only results in low product yields but also compromises the reliability of recombinant strains for biomanufacturing applications. The CRISPR/Cas9 system, renowned for its precision, efficiency, and versatility across a wide range of chassis, offers significant potential to address the limitations posed by polyploid genomes. In this study, we developed and optimized an effective sgRNA for the targeted knock-in of nucleotide sequences of varying lengths into the neutral locus slr0168 of polyploid Synechocystis using CRISPR/Cas9. The gene encoding di-geranylgeranylglycerophospholipid reductase from Sulfolobus acidocaldarius and the methyl ketone operon from Solanum habrochaites were chosen as the exemplar nucleotide sequences for incorporation into the chromosome of Synechocystis. Our results demonstrate that the designed sgRNA effectively facilitated both knock-in events and that CRISPR/Cas9 enabled complete mutant segregation in a single round of selection and induction.