Design of an effective sgRNA for CRISPR/Cas9 knock-ins in polyploidSynechocystis sp. PCC 6803

Synechocystis sp . PCC 6803 ( Synechocystis ) is a highly promising organism for the production of diverse recombinant chemicals, including biofuels. However, conventional genetic engineering in Synechocystis is challenging due to its highly polyploid genome which not only leads to low product yields but also makes the recombinant organism less reliable for use in biomanufacturing. Due to its precision, effectiveness and reliability in a vast array of chassis, CRISPR/Cas9 has the potential of overcoming the drawbacks effected by a polyploid genome. Here we identified and developed an effective sgRNA for the knock-in of nucleotide sequences of varying lengths in the neutral site slr 0168 of polyploid Synechocystis using CRISPR/Cas9. The gene encoding digeranylgeranylglycerophospholipid 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 . It is demonstrated here that our sgRNA design was effective for both knock-ins and that CRISPR/Cas9 achieves complete mutant segregation after a single step of selection and induction.