Synthetic Biology Journal ›› 2021, Vol. 2 ›› Issue (1): 106-120.DOI: 10.12211/2096-8280.2020-039
• Invited Review • Previous Articles Next Articles
Yang LI1, Xiaolin SHEN1, Xinxiao SUN1, Qipeng YUAN1, Yajun YAN2, Jia WANG1
Received:
2020-04-05
Revised:
2020-10-22
Online:
2021-03-12
Published:
2021-03-22
Contact:
Jia WANG
李洋1, 申晓林1, 孙新晓1, 袁其朋1, 闫亚军2, 王佳1
通讯作者:
王佳
作者简介:
李洋(1997—),男,硕士研究生,研究方向为光合蓝细菌合成生物学和代谢工程。 E-mail:liy97@tju.edu.cn基金资助:
CLC Number:
Yang LI, Xiaolin SHEN, Xinxiao SUN, Qipeng YUAN, Yajun YAN, Jia WANG. Advances of CRISPR gene editing in microbial synthetic biology[J]. Synthetic Biology Journal, 2021, 2(1): 106-120.
李洋, 申晓林, 孙新晓, 袁其朋, 闫亚军, 王佳. CRISPR基因编辑技术在微生物合成生物学领域的研究进展[J]. 合成生物学, 2021, 2(1): 106-120.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2020-039
Fig. 1 The cleavage and repair mechanism of CRISPR/Cas9 systems(Blue represents the recognized site of DNA, orange represents PAM sequence, and dark purple represents the recognition sequence by sgRNA. During HDR, light purple represents homologous sequences, green represents substituted sequences, orange represents RecE or Red responsible for donor DNA single strand cutting, and blue represents RecT or Red responsible for protecting sticky ends and binding to the breaking sites of genes. During NHEJ, the yellow protein represents Ku responsible for protecting the fracture site, while the orange protein is ligD responsible for connecting to the fracture site)
Fig. 2 Working mechanism of Cas12a and Cas13a systems[Orange in Cas12a represents the PAM sequence of TTTN, and blue in Cas13a represents PFS (protospacer flanking site) sequence]
物种 | 系统 | 产品 | 编辑后取得效果 | 参考文献 |
---|---|---|---|---|
E. coli | Cas9 | β-胡萝卜素 | 2.0 g/L | [ |
脂肪酸酯 | 32 g/L | [ | ||
尿苷 | 5.6 g/L | [ | ||
正丁醇 | 4.32 g/L | [ | ||
己二酸 | 68 g/L | [ | ||
庚酸 | 增强庚酸耐受性 | [ | ||
S. cerevisiae | Cas9 | 纤维二糖 | 速率提高10倍 | [ |
甲羟戊酸 | 比野生型高41倍 | [ | ||
(R,R)-(-)-2,3-丁二醇 | 12.51 g/L | [ | ||
β-胡萝卜素 | 提高了3倍 | [ | ||
紫杉二烯 | 提高了25倍 | [ | ||
乙醇 | 提高了74.7% | [ | ||
脂肪酸 | 提高了30倍 | [ | ||
3-羟基丙酸 | 11.4 g/L | [ | ||
萜类 | 未具体说明 | [ | ||
青蒿素类 | 740 mg/L | [ | ||
Actinoplanes sp. | Cas9 | 阿卡波糖 | 提高了纯度 | [ |
Streptomyces rimosus | Cas9 | 土霉素 | 增加了36.8% | [ |
Saccharopolyspora erythraea | Cas9 | 红霉素 | 比野生型高80.3% | [ |
Corynebacterium glutamicum | Cas12a | 半胱氨酸;丝氨酸 | 半胱氨酸提高3.7倍,丝氨酸提高2.5倍 | [ |
Tab. 1 Applications of CRISPR-based technologies for the construction of microbial cell factories
物种 | 系统 | 产品 | 编辑后取得效果 | 参考文献 |
---|---|---|---|---|
E. coli | Cas9 | β-胡萝卜素 | 2.0 g/L | [ |
脂肪酸酯 | 32 g/L | [ | ||
尿苷 | 5.6 g/L | [ | ||
正丁醇 | 4.32 g/L | [ | ||
己二酸 | 68 g/L | [ | ||
庚酸 | 增强庚酸耐受性 | [ | ||
S. cerevisiae | Cas9 | 纤维二糖 | 速率提高10倍 | [ |
甲羟戊酸 | 比野生型高41倍 | [ | ||
(R,R)-(-)-2,3-丁二醇 | 12.51 g/L | [ | ||
β-胡萝卜素 | 提高了3倍 | [ | ||
紫杉二烯 | 提高了25倍 | [ | ||
乙醇 | 提高了74.7% | [ | ||
脂肪酸 | 提高了30倍 | [ | ||
3-羟基丙酸 | 11.4 g/L | [ | ||
萜类 | 未具体说明 | [ | ||
青蒿素类 | 740 mg/L | [ | ||
Actinoplanes sp. | Cas9 | 阿卡波糖 | 提高了纯度 | [ |
Streptomyces rimosus | Cas9 | 土霉素 | 增加了36.8% | [ |
Saccharopolyspora erythraea | Cas9 | 红霉素 | 比野生型高80.3% | [ |
Corynebacterium glutamicum | Cas12a | 半胱氨酸;丝氨酸 | 半胱氨酸提高3.7倍,丝氨酸提高2.5倍 | [ |
技术 | 特点 | 优势 | 缺陷 | |||||
---|---|---|---|---|---|---|---|---|
类型 | 标靶 | 是否需要tracrRNA | PAM | 间隔区间长度 | 切割结构域 | |||
CRISPR/Cas9 | II | dsDNA | 是 | NGG-3' | 20 | HNH和RuvC | 成本低;打靶效率高;操作容易;应用广泛 | PAM(NGG)依赖性;依然存在脱靶效应;可控性差;某些物种编辑效率低;内源性系统的干扰;对细胞致死性 |
CRISPR/Cas12a(Cpf1) | V | dsDNA | 否 | 5'-TTTN | 23 | RuvC | 切割产生黏性末端;低毒性;蛋白分子较小;在某些物种中编辑效率高于Cas9系统;不需要tracrRNA | 研究不成熟 |
CRISPR/Cas13a(C2c2) | VI | ssRNA | 否 | PFS | — | HEPN | 对RNA干扰和编辑;脱靶率低于Cas9系统;蛋白分子小 | 研究较少;应用领域目前较窄 |
Tab. 2 The comparison and summary of CRISPR/Cas9, Cas12a and Cas13a
技术 | 特点 | 优势 | 缺陷 | |||||
---|---|---|---|---|---|---|---|---|
类型 | 标靶 | 是否需要tracrRNA | PAM | 间隔区间长度 | 切割结构域 | |||
CRISPR/Cas9 | II | dsDNA | 是 | NGG-3' | 20 | HNH和RuvC | 成本低;打靶效率高;操作容易;应用广泛 | PAM(NGG)依赖性;依然存在脱靶效应;可控性差;某些物种编辑效率低;内源性系统的干扰;对细胞致死性 |
CRISPR/Cas12a(Cpf1) | V | dsDNA | 否 | 5'-TTTN | 23 | RuvC | 切割产生黏性末端;低毒性;蛋白分子较小;在某些物种中编辑效率高于Cas9系统;不需要tracrRNA | 研究不成熟 |
CRISPR/Cas13a(C2c2) | VI | ssRNA | 否 | PFS | — | HEPN | 对RNA干扰和编辑;脱靶率低于Cas9系统;蛋白分子小 | 研究较少;应用领域目前较窄 |
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