Design of an Effective sgRNA for CRISPR/Cas9 Knock-Ins and Full Mutant Segregation in Polyploid <i>Synechocystis</i> sp. PCC 6803
<i>Synechocystis</i> sp. PCC 6803 is a highly promising organism for the production of diverse recombinant compounds, including biofuels. However, conventional genetic engineering in <i>Synechocystis</i> presents challenges due to its highly polyploid genome, which not only r...
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| Main Authors: | , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-01-01
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| Series: | SynBio |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2674-0583/3/1/3 |
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| Summary: | <i>Synechocystis</i> sp. PCC 6803 is a highly promising organism for the production of diverse recombinant compounds, including biofuels. However, conventional genetic engineering in <i>Synechocystis</i> 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 <i>slr</i>0168 of polyploid <i>Synechocystis</i> using CRISPR/Cas9. The gene encoding di-geranylgeranylglycerophospholipid reductase from <i>Sulfolobus acidocaldarius</i> and the methyl ketone operon from <i>Solanum habrochaites</i> were chosen as the exemplar nucleotide sequences for incorporation into the chromosome of <i>Synechocystis.</i> 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. |
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| ISSN: | 2674-0583 |