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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2025-01-01
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| author | Maria Isabel Nares-Rodriguez Esther Karunakaran |
| author_facet | Maria Isabel Nares-Rodriguez Esther Karunakaran |
| author_sort | Maria Isabel Nares-Rodriguez |
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| description | <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. |
| format | Article |
| id | doaj-art-b5b23a2ed8304b60ac80668d3d108a2d |
| institution | Kabale University |
| issn | 2674-0583 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
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| series | SynBio |
| spelling | doaj-art-b5b23a2ed8304b60ac80668d3d108a2d2025-08-20T03:43:51ZengMDPI AGSynBio2674-05832025-01-0131310.3390/synbio3010003Design of an Effective sgRNA for CRISPR/Cas9 Knock-Ins and Full Mutant Segregation in Polyploid <i>Synechocystis</i> sp. PCC 6803Maria Isabel Nares-Rodriguez0Esther Karunakaran1School of Chemical, Materials and Biological Engineering, University of Sheffield, Sheffield S1 3JD, UKSchool of Chemical, Materials and Biological Engineering, University of Sheffield, Sheffield S1 3JD, UK<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.https://www.mdpi.com/2674-0583/3/1/3CRISPR/Cas9sgRNA designfull mutant segregation<i>Synechocystis</i> sp. PCC 6803single induction step |
| spellingShingle | Maria Isabel Nares-Rodriguez Esther Karunakaran Design of an Effective sgRNA for CRISPR/Cas9 Knock-Ins and Full Mutant Segregation in Polyploid <i>Synechocystis</i> sp. PCC 6803 SynBio CRISPR/Cas9 sgRNA design full mutant segregation <i>Synechocystis</i> sp. PCC 6803 single induction step |
| title | Design of an Effective sgRNA for CRISPR/Cas9 Knock-Ins and Full Mutant Segregation in Polyploid <i>Synechocystis</i> sp. PCC 6803 |
| title_full | Design of an Effective sgRNA for CRISPR/Cas9 Knock-Ins and Full Mutant Segregation in Polyploid <i>Synechocystis</i> sp. PCC 6803 |
| title_fullStr | Design of an Effective sgRNA for CRISPR/Cas9 Knock-Ins and Full Mutant Segregation in Polyploid <i>Synechocystis</i> sp. PCC 6803 |
| title_full_unstemmed | Design of an Effective sgRNA for CRISPR/Cas9 Knock-Ins and Full Mutant Segregation in Polyploid <i>Synechocystis</i> sp. PCC 6803 |
| title_short | Design of an Effective sgRNA for CRISPR/Cas9 Knock-Ins and Full Mutant Segregation in Polyploid <i>Synechocystis</i> sp. PCC 6803 |
| title_sort | design of an effective sgrna for crispr cas9 knock ins and full mutant segregation in polyploid i synechocystis i sp pcc 6803 |
| topic | CRISPR/Cas9 sgRNA design full mutant segregation <i>Synechocystis</i> sp. PCC 6803 single induction step |
| url | https://www.mdpi.com/2674-0583/3/1/3 |
| work_keys_str_mv | AT mariaisabelnaresrodriguez designofaneffectivesgrnaforcrisprcas9knockinsandfullmutantsegregationinpolyploidisynechocystisisppcc6803 AT estherkarunakaran designofaneffectivesgrnaforcrisprcas9knockinsandfullmutantsegregationinpolyploidisynechocystisisppcc6803 |