Synthetic Biology Journal ›› 2023, Vol. 4 ›› Issue (4): 824-839.DOI: 10.12211/2096-8280.2022-077
• Research Article • Previous Articles Next Articles
Tiantian WANG1,2, Hong ZHU1, Chen YANG1
Received:
2022-12-30
Revised:
2023-02-20
Online:
2023-09-14
Published:
2023-08-31
Contact:
Chen YANG
王甜甜1,2, 朱虹1, 杨琛1
通讯作者:
杨琛
作者简介:
基金资助:
CLC Number:
Tiantian WANG, Hong ZHU, Chen YANG. Development of CRISPRa for metabolic engineering applications in cyanobacteria[J]. Synthetic Biology Journal, 2023, 4(4): 824-839.
王甜甜, 朱虹, 杨琛. 蓝细菌CRISPRa系统的开发及其代谢工程应用[J]. 合成生物学, 2023, 4(4): 824-839.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2022-077
引物 Primer | 5′-3′序列 Sequences 5′-3′ |
---|---|
Pendo-F | GAGACACAACGTGGCTTTCCCGCGGCCGCTTACGAAATCATCCTGTGGAGCTTAGTAG |
Pendo-R | CTATTGAGTATTTCTTATCCATTTTTGCCTCCTAAAATAAAAAGTTTAAATTAAATC |
dCas9-F | ATGGATAAGAAATACTCAATAGGCTTAG |
dCas9-R | AATGATTTTCTGGTGGCTCATGGAACCGCCACCGCCGGAACCGCCACCGCCTGCGCAGTCACCTCCTAGCTGACTCAA |
nudⅠ-F | ATGCGACAACGGACTATTGTATG |
nudⅠ-R | GATGGGTAATTTCGCTGAGATGCATGGTATATCTCCTTCTTACTAGTCTCTCTCTTGTACATTACAGAAGACCTTTCAAACGTAAC |
dxs-F | ATGCATCTCAGCGAAATTACCCATC |
dxs-R | AGCCGGATTAATAATCTGGCTTTTTATATTCTCTTTAAGCCGAAGCAGCACCAAT |
P18082-F | TTAATTAACCTGCCGAG |
P18082-R | AGCTCTTCGCCCTTGCTCATGGTACCTTTCTCCTCTTTAATGAATTCGCCTG |
P18082 upstream-F | ACCGTTTCAGCTGGTGATTTGGATCCGCATGCCCGATCAACGTCTC |
P18082 upstream -R | ACCTCGGCAGGTTAATTAAGACCGGTATGCCTAATGTA |
sfgfp -F | ATGAGCAAGGGCGAAGAG |
sfgfp-R | AGCCAAGCTGGAGACCGTTTAAACTCACTACTTGTACAGTTCATCCATGCCA |
qPCR-nudI-F | ATGGTGCTTATTTGCT |
qPCR-nudI-R | TGTTCTCCCAGTTCTT |
qPCR-dxs-F | ACCCATCCCAACCAGC |
qPCR-dxs-R | TTCCACCACGCCCAAG |
qPCR-gpps-F | GCGGGTGGAACGGCTG |
qPCR-gpps-R | CCTTGTGATTGGTGGG |
NSⅡ-up-F验证 | ACCTTGCGTCGGTGCTGAGTC |
dxs-R验证 | TTGCTCAAGCTGAGCAACCGAC |
NSⅢ-up-F验证 | GATGCACGAGCGTAATGCTCAC |
NSⅢ-del-R验证 | TCTCGCTAATTGTGGGAGAGGAG |
Table 3 Primers used in this study
引物 Primer | 5′-3′序列 Sequences 5′-3′ |
---|---|
Pendo-F | GAGACACAACGTGGCTTTCCCGCGGCCGCTTACGAAATCATCCTGTGGAGCTTAGTAG |
Pendo-R | CTATTGAGTATTTCTTATCCATTTTTGCCTCCTAAAATAAAAAGTTTAAATTAAATC |
dCas9-F | ATGGATAAGAAATACTCAATAGGCTTAG |
dCas9-R | AATGATTTTCTGGTGGCTCATGGAACCGCCACCGCCGGAACCGCCACCGCCTGCGCAGTCACCTCCTAGCTGACTCAA |
nudⅠ-F | ATGCGACAACGGACTATTGTATG |
nudⅠ-R | GATGGGTAATTTCGCTGAGATGCATGGTATATCTCCTTCTTACTAGTCTCTCTCTTGTACATTACAGAAGACCTTTCAAACGTAAC |
dxs-F | ATGCATCTCAGCGAAATTACCCATC |
dxs-R | AGCCGGATTAATAATCTGGCTTTTTATATTCTCTTTAAGCCGAAGCAGCACCAAT |
P18082-F | TTAATTAACCTGCCGAG |
P18082-R | AGCTCTTCGCCCTTGCTCATGGTACCTTTCTCCTCTTTAATGAATTCGCCTG |
P18082 upstream-F | ACCGTTTCAGCTGGTGATTTGGATCCGCATGCCCGATCAACGTCTC |
P18082 upstream -R | ACCTCGGCAGGTTAATTAAGACCGGTATGCCTAATGTA |
sfgfp -F | ATGAGCAAGGGCGAAGAG |
sfgfp-R | AGCCAAGCTGGAGACCGTTTAAACTCACTACTTGTACAGTTCATCCATGCCA |
qPCR-nudI-F | ATGGTGCTTATTTGCT |
qPCR-nudI-R | TGTTCTCCCAGTTCTT |
qPCR-dxs-F | ACCCATCCCAACCAGC |
qPCR-dxs-R | TTCCACCACGCCCAAG |
qPCR-gpps-F | GCGGGTGGAACGGCTG |
qPCR-gpps-R | CCTTGTGATTGGTGGG |
NSⅡ-up-F验证 | ACCTTGCGTCGGTGCTGAGTC |
dxs-R验证 | TTGCTCAAGCTGAGCAACCGAC |
NSⅢ-up-F验证 | GATGCACGAGCGTAATGCTCAC |
NSⅢ-del-R验证 | TCTCGCTAATTGTGGGAGAGGAG |
菌株 Strain | 基因型 Genotype | 来源 Source |
---|---|---|
Synechococcus elongatus PCC7942 | Wild type | ATCC |
PCC 7942-G01 | P18082 sfgfp integrated at NSⅡ | This work |
PCC 7942-G02 | P18082 sfgfp pcpc sgRNA 106 integrated at NSⅡ | This work |
PCC 7942-G03 | P18082 sfgfp pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G09 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G10 | Pendo dCas9-CAGGGGSGGGGS-rpoA integrated at NSⅢ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G11 | Pendo dCas9-CAGGGGSGGGGS-rpoD integrated at NSⅡ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G12 | Pendo dCas9-CAGGGGSGGGGS-rpoA NTD integrated at NSⅢ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G13 | Pendo dCas9-CAGGGGSGGGGS-soxS integrated at NSⅢ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G14 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfp Pcpc sgRNA H1 integrated at NSⅡ | This work |
PCC 7942-G15 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfp Pcpc sgRNA H2 integrated at NSⅡ | This work |
PCC 7942-G16 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfp Pcpc sgRNA H3 integrated at NSⅡ | This work |
PCC 7942-G17 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfp Pcpc sgRNA H5 integrated at NSⅡ | This work |
PCC 7942-G18 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ | This work |
PCC 7942-G19 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G27 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H1 integrated at NSⅡ | This work |
PCC 7942-G28 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H2 integrated at NSⅡ | This work |
PCC 7942-G29 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H3 integrated at NSⅡ | This work |
PCC 7942-G30 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H5 integrated at NSⅡ | This work |
PCC 7942-G31 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H1 integrated at NSⅡ | This work |
PCC 7942-G32 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H2 integrated at NSⅡ | This work |
PCC 7942-G33 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H3 integrated at NSⅡ | This work |
PCC 7942-G34 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G35 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H5 integrated at NSⅡ | This work |
PCC 7942-G36 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H1 integrated at NSⅡ | This work |
PCC 7942-G37 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H2 integrated at NSⅡ | This work |
PCC 7942-G38 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H3 integrated at NSⅡ | This work |
PCC 7942-G39 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G40 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H5 integrated at NSⅡ | This work |
PCC 7942-G45 | PJ23108 dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ | This work |
PCC 7942-S01 | P18082 nudⅠ Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-S02 | P18082 nudⅠ dxs Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-S03 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 nudⅠ Pcpc sgRNA-H4 integrated at NSⅡ | This work |
PCC 7942-S04 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ Pcpc sgRNA-H4 integrated at NSⅡ | This work |
PCC 7942-S05 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 nudⅠ dxs Pcpc sgRNA-H4 integrated at NSⅡ | This work |
PCC 7942-S06 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-H4 integrated at NSⅡ | This work |
PCC 7942-S07 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-106 integrated at NSⅡ | This work |
PCC 7942-S12 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-H4 Ptrc crRNA-g1 PJ23119 tracrRNA integrated at NSⅡ | This work |
PCC 7942-S13 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-H4 Ptrc crRNA-g2 PJ23119 tracrRNA integrated at NSⅡ | This work |
PCC 7942-S14 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-H4 Ptrc crRNA-106 PJ23119 tracrRNA integrated at NSⅡ | This work |
Table 1 Strains used in this study
菌株 Strain | 基因型 Genotype | 来源 Source |
---|---|---|
Synechococcus elongatus PCC7942 | Wild type | ATCC |
PCC 7942-G01 | P18082 sfgfp integrated at NSⅡ | This work |
PCC 7942-G02 | P18082 sfgfp pcpc sgRNA 106 integrated at NSⅡ | This work |
PCC 7942-G03 | P18082 sfgfp pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G09 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G10 | Pendo dCas9-CAGGGGSGGGGS-rpoA integrated at NSⅢ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G11 | Pendo dCas9-CAGGGGSGGGGS-rpoD integrated at NSⅡ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G12 | Pendo dCas9-CAGGGGSGGGGS-rpoA NTD integrated at NSⅢ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G13 | Pendo dCas9-CAGGGGSGGGGS-soxS integrated at NSⅢ P18082 sfgfpPcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G14 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfp Pcpc sgRNA H1 integrated at NSⅡ | This work |
PCC 7942-G15 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfp Pcpc sgRNA H2 integrated at NSⅡ | This work |
PCC 7942-G16 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfp Pcpc sgRNA H3 integrated at NSⅡ | This work |
PCC 7942-G17 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 sfgfp Pcpc sgRNA H5 integrated at NSⅡ | This work |
PCC 7942-G18 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ | This work |
PCC 7942-G19 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G27 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H1 integrated at NSⅡ | This work |
PCC 7942-G28 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H2 integrated at NSⅡ | This work |
PCC 7942-G29 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H3 integrated at NSⅡ | This work |
PCC 7942-G30 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H5 integrated at NSⅡ | This work |
PCC 7942-G31 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H1 integrated at NSⅡ | This work |
PCC 7942-G32 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H2 integrated at NSⅡ | This work |
PCC 7942-G33 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H3 integrated at NSⅡ | This work |
PCC 7942-G34 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G35 | Pendo rpoZ-CAGGGGSGGGGS-dCas9 integrated at rpoZ P18082 sfgfp Pcpc sgRNA H5 integrated at NSⅡ | This work |
PCC 7942-G36 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H1 integrated at NSⅡ | This work |
PCC 7942-G37 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H2 integrated at NSⅡ | This work |
PCC 7942-G38 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H3 integrated at NSⅡ | This work |
PCC 7942-G39 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-G40 | Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 sfgfp Pcpc sgRNA H5 integrated at NSⅡ | This work |
PCC 7942-G45 | PJ23108 dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ | This work |
PCC 7942-S01 | P18082 nudⅠ Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-S02 | P18082 nudⅠ dxs Pcpc sgRNA H4 integrated at NSⅡ | This work |
PCC 7942-S03 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 nudⅠ Pcpc sgRNA-H4 integrated at NSⅡ | This work |
PCC 7942-S04 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ Pcpc sgRNA-H4 integrated at NSⅡ | This work |
PCC 7942-S05 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at NSⅢ P18082 nudⅠ dxs Pcpc sgRNA-H4 integrated at NSⅡ | This work |
PCC 7942-S06 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-H4 integrated at NSⅡ | This work |
PCC 7942-S07 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-106 integrated at NSⅡ | This work |
PCC 7942-S12 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-H4 Ptrc crRNA-g1 PJ23119 tracrRNA integrated at NSⅡ | This work |
PCC 7942-S13 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-H4 Ptrc crRNA-g2 PJ23119 tracrRNA integrated at NSⅡ | This work |
PCC 7942-S14 | Pendo dCas9-CAGGGGSGGGGS-rpoZ integrated at rpoZ P18082 nudⅠ dxs Pcpc sgRNA-H4 Ptrc crRNA-106 PJ23119 tracrRNA integrated at NSⅡ | This work |
质粒 Plasmid | 描述 Description | 来源 Source |
---|---|---|
P01 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp | This work |
P02 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H1 | This work |
P03 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H2 | This work |
P04 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H3 | This work |
P05 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H4 | This work |
P06 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H5 | This work |
P07 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA 106 | This work |
P08 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ Pcpc sgRNA H4 | This work |
P09 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc sgRNA H4 | This work |
P10 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc sgRNA 106 | This work |
P11 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc crRNA g1 PJ23119 tracrRNA | This work |
P12 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc crRNA g2 PJ23119 tracrRNA | This work |
P13 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc crRNA 106 PJ23119 tracrRNA | This work |
P14 | PCL1920; Kanr ; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-soxS | This work |
P15 | PCL1920; Kanr; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoZ | This work |
P16 | PCL1920; Kanr; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoD | This work |
P17 | PCL1920; Kanr; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoA | This work |
P18 | PCL1920; Kanr; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoA NTD | This work |
P19 | PCL1920; Kanr; RpoZ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoZ | This work |
P20 | PCL1920; Kanr;RpoZ targeting; Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ | This work |
P21 | PCL1920; Kanr; RpoZ targeting; Pendo rpoZ-CAGGGGSGGGGS-dCas9 | This work |
P22 | PCL1920; Kanr; RpoZ targeting; PJ23108 dCas9-CAGGGGSGGGGS-rpoZ | This work |
P23 | PCL1920; Kanr; RpoZ targeting; Pcpc dCas9-CAGGGGSGGGGS-rpoZ | This work |
P24 | PCL1920; Kanr; RpoZ targeting; Pcpc dCas9 | This work |
P25 | PCL1920; Kanr; NSⅢ targeting; Pcpc dCas9-CAGGGGSGGGGS-rpoZ | This work |
Table 2 Plasmids used in this study
质粒 Plasmid | 描述 Description | 来源 Source |
---|---|---|
P01 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp | This work |
P02 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H1 | This work |
P03 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H2 | This work |
P04 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H3 | This work |
P05 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H4 | This work |
P06 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA H5 | This work |
P07 | PCL1920; Specr; NSⅡ targeting; P18082 sfgfp Pcpc sgRNA 106 | This work |
P08 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ Pcpc sgRNA H4 | This work |
P09 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc sgRNA H4 | This work |
P10 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc sgRNA 106 | This work |
P11 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc crRNA g1 PJ23119 tracrRNA | This work |
P12 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc crRNA g2 PJ23119 tracrRNA | This work |
P13 | PCL1920; Specr; NSⅡ targeting; P18082 nudⅠ dxs Pcpc crRNA 106 PJ23119 tracrRNA | This work |
P14 | PCL1920; Kanr ; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-soxS | This work |
P15 | PCL1920; Kanr; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoZ | This work |
P16 | PCL1920; Kanr; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoD | This work |
P17 | PCL1920; Kanr; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoA | This work |
P18 | PCL1920; Kanr; NSⅢ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoA NTD | This work |
P19 | PCL1920; Kanr; RpoZ targeting; Pendo dCas9-CAGGGGSGGGGS-rpoZ | This work |
P20 | PCL1920; Kanr;RpoZ targeting; Pendo rpoZ-CAGGGGSGGGGS-dCas9-CAGGGGSGGGGS-rpoZ | This work |
P21 | PCL1920; Kanr; RpoZ targeting; Pendo rpoZ-CAGGGGSGGGGS-dCas9 | This work |
P22 | PCL1920; Kanr; RpoZ targeting; PJ23108 dCas9-CAGGGGSGGGGS-rpoZ | This work |
P23 | PCL1920; Kanr; RpoZ targeting; Pcpc dCas9-CAGGGGSGGGGS-rpoZ | This work |
P24 | PCL1920; Kanr; RpoZ targeting; Pcpc dCas9 | This work |
P25 | PCL1920; Kanr; NSⅢ targeting; Pcpc dCas9-CAGGGGSGGGGS-rpoZ | This work |
Fig. 1 Configuration of CRISPRa in PCC7942(a) Components of the CRISPRa system. (b) The expression cassette encoding dCas9 anda transcriptional activator fusion protein was inserted into the NSⅢ site of PCC7942, and the report genes sfgfp and sgRNA were integrated into the NSⅡ site of PCC7942. (c) The fluorescence intensities of strains G03, G09, G10, G11, G12, and G13 with different transcriptional activators. (d) The fluorescence signal level of G01(control strain without sgRNA) and G02(control strain with an off target sgRNA 106).
靶点 TargetSites | DNA序列(5′→3′) DNA Sequences(5′→3′) | Target Strand | Distance toTSS/bp |
---|---|---|---|
H1 | ATGTAACACCGTGCGTGTTG | NT | -216 |
H2 | GAAGATCCGGCCTGCAGCCA | NT | -236 |
H3 | GGCTCGAGTCGACAGTTCAT | NT | -273 |
H4 | CTACGGAACTCTTGTGCGTA | T | -327 |
H5 | GCAAAAGCTCATTTCTGAAG | T | -397 |
g1 | CACTCAAAGGATAGACGGGA | NT | +48 |
g2 | TCCCATTGCGCTAAGCCCTA | NT | +960 |
Table 4 Targeting sequences for the CRISPRa system used in this study
靶点 TargetSites | DNA序列(5′→3′) DNA Sequences(5′→3′) | Target Strand | Distance toTSS/bp |
---|---|---|---|
H1 | ATGTAACACCGTGCGTGTTG | NT | -216 |
H2 | GAAGATCCGGCCTGCAGCCA | NT | -236 |
H3 | GGCTCGAGTCGACAGTTCAT | NT | -273 |
H4 | CTACGGAACTCTTGTGCGTA | T | -327 |
H5 | GCAAAAGCTCATTTCTGAAG | T | -397 |
g1 | CACTCAAAGGATAGACGGGA | NT | +48 |
g2 | TCCCATTGCGCTAAGCCCTA | NT | +960 |
Fig. 2 Optimization of the CRISPRa system(a)Scheme of different targeting sites for CRISPRa. (b) The fluorescence intensities of strains G09, G14, G15,G16, and G17 with different targeting sites. (c) The fluorescence intensity of G19 strain with genetic knockout of endogenous rpoZ. (d) Effect of different orientations of the dCas9 and RpoZ fusion on CRISPRa performance. (e) Expression of dCas9-RpoZ was increased in G45 strain by replacing the promoter Pendo with PJ23108. (f) The fluorescence intensity of G45 strain.
Fig. 3 Activation of nudⅠ by CRISPRa(a) Scheme of nudⅠ activation by CRISPRa. The sgRNA H4 and nudⅠ were inserted into the NSⅡ site. The DNA fragment encoding dCas9-RpoZ was inserted into the NSⅢ site or to replace endogenous rpoZ, generating strains S03 and S04, respectively. The control strain S01 lacked dCas9-RpoZ. (b) Isopentenol production by strains S01, S03, and S04. Cells were cultivated for 96 h, and isopentenol in the culture supernatant was detected by a colorimetric assay. (c) Transcriptional level of nudⅠ in strains S01, S03, and S04. RNA was isolated from cellscultivated for 72 h.
Fig. 4 Simultaneous activation of nudⅠ and dxs by CRISPRa(a) 1-Deoxy-D-xylulose 5-phosphate synthase encoded by dxs catalyzes the first reaction of the MEP pathway. (b)Scheme of nudⅠ and dxs activation by CRISPRa. The sgRNA H4, nudⅠ, and dxs were inserted into the NSⅡ site. The DNA fragment encoding dCas9-RpoZ was inserted into the NSⅢ site or to replace endogenous rpoZ, generating strains S05 and S06, respectively. S02 (without dCas9-RpoZ) and S07 (with an off target sgRNA 106) are the control strains. (c) Isopentenol production by strains S02, S05, S06, and S07.Cells were cultivated for 96 h, and isopentenol in the culture supernatant was detected by a colorimetric assay. (f) Transcriptional levels of nudI and dxs in strains S02, S05, S06, and S07. RNA was isolated from cells cultivated for 72 h.
Fig. 5 Simultaneous activation of nudⅠ and dxs and repression of gpps by CRISPRa(a) gpps catalyzes a key competing reaction for isopentenol biosynthesis; (b) Location of the gpps gene and the targeting sites g1 and g2 on the PCC 7942 genome; (c) The g1 and g2 sites of gpps in strain S06 were targeted, generating strains S12 and S13, respectively. S14 (with an off target sgRNA 106) is a control strain. (d) Transcriptional levels of gpps, nudⅠ and dxs in strains S02, S06, S12, S13, and S14. RNA was isolated from cells grown to OD730 of about 0.7. (e) Isopentenol production by strains S02, S06, S13, and S14. Isopentenol in the culture supernatant was detected by a colorimetric assay. (f) Isopentenol production by strains S02, S06, and S13. Culture supernatants were collected at OD730 of about 2.0, and isopentenol was detected by gas chromatography.
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