Synthetic Biology Journal ›› 2022, Vol. 3 ›› Issue (6): 1201-1217.DOI: 10.12211/2096-8280.2022-041
• Invited Review • Previous Articles Next Articles
Jiaxin LIU1, Chi CHENG1,2, Xinqi LI1, Chaojun WANG2, Ying ZHANG2, Chuang XUE1,2
Received:
2022-07-18
Revised:
2022-08-30
Online:
2023-01-17
Published:
2022-12-31
Contact:
Chi CHENG, Chuang XUE
刘佳昕1, 程驰1,2, 李欣启1, 汪超俊2, 张颖2, 薛闯1,2
通讯作者:
程驰,薛闯
作者简介:
基金资助:
Jiaxin LIU, Chi CHENG, Xinqi LI, Chaojun WANG, Ying ZHANG, Chuang XUE. Recent progress in the molecular genetic modification tools of Clostridium[J]. Synthetic Biology Journal, 2022, 3(6): 1201-1217.
刘佳昕, 程驰, 李欣启, 汪超俊, 张颖, 薛闯. 梭菌分子遗传改造工具研究进展[J]. 合成生物学, 2022, 3(6): 1201-1217.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2022-041
遗传操作工具 | 优点 | 缺点 | 梭菌菌株 | 基因 | 文献 |
---|---|---|---|---|---|
基于游离质粒的基因过表达 | 设计简单,易于操作 | 质粒不稳定,需靠抗生素维持 | C. acetobutylicum ATCC 824 C. acetobutylicum DSM 792 C. paraputrificum M-21 C. tyrobutyricum JM1 C. tyrobutyricum ATCC 25755 C. perfringens SM101 C. cellulolyticum H10 | spo0A, hydA, adhE2, adC, groESL, txeR, tcdC, pdc-adhII | [ |
反义RNA技术 | 致死率低,易于筛选 | 仅在转录水平调控 | C. acetobutylicum ATCC 824 | ptb, buk, CoAT | [ |
转座子突变技术 | 可用于建立突变体库 | 难以控制插入位点 | C. difficile CD37 C. perfringens Strain 13 | random | [ |
二型内含子技术 | 操作简单,几乎适用于所有梭菌 | 脱靶概率较大,存在极性效应 | C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052 | glcG, cbei2385, xylR, bdhA, bdhB, ptb, ack, adc | [ |
同源重组 | 可精确进行基因编辑 | 同源重组效率很低 | C. acetobutylicum NCIMB 8052 | gutD, spo0A | [ |
反筛标记介导的等位基因替换 | 相比纯粹的等位基因替换,效率有所提升 | 受制于第一次单交换效率 | C. acetobutylicum ATCC 824 | adh | [ |
Ⅰ-SceⅠ归巢内切酶介导的等位基因替换 | 适用范围广,在革兰氏阳性和阴性菌中均可使用 | 设计复杂,操作烦琐,周期长 | C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052 | adc, glcG, xylR | [ |
噬菌体丝氨酸重组酶介导的位点特异性基因编辑技术 | 适用于大片段DNA快速整合到宿主染色体上 | 受限于附着位点attb/p,应用范围小 | C. ljungdahlii DSM 13528 | thl, crt, bcd, etfB, etfA, hbd, ptb, buk | [ |
Red/ET重组酶介导的同源重组 | 所需同源臂较短、不受限制性酶切位点限制 | 尚不完善 | C. acetobutylicum SMB009 | ermC | [ |
CRISPR/Cas9系统介导的基因编辑 | 提高同源重组效率,可实现精确编辑 | 毒性大,很难获得转化子 | C. acetobutylicum DSM792 C. saccharoperbutylacetonicum N1-4 C. cellulovorans C. autoethanogenum DSM10061 | hprK, cac1502, pta, buk, clocel2243, adhE1, ctf,pyrE, 2,3-bdh | [ |
CRISPR/Cas9n系统介导的基因编辑 | 相比于CRISPR/Cas9毒性有所降低,转化子数目增加 | 仍有毒性,转化子依然偏少 | C. cellulolyticum H10 C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052 | pyrF, spo0A | [ |
CRISPR/dCas9系统介导的基因表达下调 | 相比于CRISPR/Cas9毒性大大降低,易于得到转化子 | 依赖于sgRNA和基因;仅在转录水平调控 | C. pasteurianum ATCC 6013 C. acetobutylicum DSM792 | hprK, glpX | [ |
Tab. 1 Comparison of different genetic tools applicable in Clostridium
遗传操作工具 | 优点 | 缺点 | 梭菌菌株 | 基因 | 文献 |
---|---|---|---|---|---|
基于游离质粒的基因过表达 | 设计简单,易于操作 | 质粒不稳定,需靠抗生素维持 | C. acetobutylicum ATCC 824 C. acetobutylicum DSM 792 C. paraputrificum M-21 C. tyrobutyricum JM1 C. tyrobutyricum ATCC 25755 C. perfringens SM101 C. cellulolyticum H10 | spo0A, hydA, adhE2, adC, groESL, txeR, tcdC, pdc-adhII | [ |
反义RNA技术 | 致死率低,易于筛选 | 仅在转录水平调控 | C. acetobutylicum ATCC 824 | ptb, buk, CoAT | [ |
转座子突变技术 | 可用于建立突变体库 | 难以控制插入位点 | C. difficile CD37 C. perfringens Strain 13 | random | [ |
二型内含子技术 | 操作简单,几乎适用于所有梭菌 | 脱靶概率较大,存在极性效应 | C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052 | glcG, cbei2385, xylR, bdhA, bdhB, ptb, ack, adc | [ |
同源重组 | 可精确进行基因编辑 | 同源重组效率很低 | C. acetobutylicum NCIMB 8052 | gutD, spo0A | [ |
反筛标记介导的等位基因替换 | 相比纯粹的等位基因替换,效率有所提升 | 受制于第一次单交换效率 | C. acetobutylicum ATCC 824 | adh | [ |
Ⅰ-SceⅠ归巢内切酶介导的等位基因替换 | 适用范围广,在革兰氏阳性和阴性菌中均可使用 | 设计复杂,操作烦琐,周期长 | C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052 | adc, glcG, xylR | [ |
噬菌体丝氨酸重组酶介导的位点特异性基因编辑技术 | 适用于大片段DNA快速整合到宿主染色体上 | 受限于附着位点attb/p,应用范围小 | C. ljungdahlii DSM 13528 | thl, crt, bcd, etfB, etfA, hbd, ptb, buk | [ |
Red/ET重组酶介导的同源重组 | 所需同源臂较短、不受限制性酶切位点限制 | 尚不完善 | C. acetobutylicum SMB009 | ermC | [ |
CRISPR/Cas9系统介导的基因编辑 | 提高同源重组效率,可实现精确编辑 | 毒性大,很难获得转化子 | C. acetobutylicum DSM792 C. saccharoperbutylacetonicum N1-4 C. cellulovorans C. autoethanogenum DSM10061 | hprK, cac1502, pta, buk, clocel2243, adhE1, ctf,pyrE, 2,3-bdh | [ |
CRISPR/Cas9n系统介导的基因编辑 | 相比于CRISPR/Cas9毒性有所降低,转化子数目增加 | 仍有毒性,转化子依然偏少 | C. cellulolyticum H10 C. acetobutylicum ATCC 824 C. beijerinckii NCIMB 8052 | pyrF, spo0A | [ |
CRISPR/dCas9系统介导的基因表达下调 | 相比于CRISPR/Cas9毒性大大降低,易于得到转化子 | 依赖于sgRNA和基因;仅在转录水平调控 | C. pasteurianum ATCC 6013 C. acetobutylicum DSM792 | hprK, glpX | [ |
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