论文
论文标题: Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120
作者: Niu, Tian-Cai; Lin, Gui-Ming; Xie, Li-Rui; Wang, Zi-Qian; Xing, Wei-Yue; Zhang, Ju-Yuan; Zhang, Cheng-Cai
出版刊物: ACS SYNTHETIC BIOLOGY
出版日期: JAN
出版年份: 2019
卷/期: 1
DOI: 10.1021/acssynbio.8b00437
论文摘要: CRISPR systems, such as CRISPR-Cas9 and CRISPR-Cpf1, have been successfully used for genome editing in a variety of organisms. Although the technique of CRISPR-Cpf1 has been applied in cyanobacteria recently, its use was limited without exploiting the full potential of such a powerful genetic system. Using the cyanobacterium Anabaena PCC 7120 as a model strain, we improved the tools and designed genetic strategies based on CRISPR-Cpf1, which enabled us to realize genetic experiments that have been so far difficult to do in cyanobacteria. The development includes: (1) a two-spacers strategy for single genomic modification, with a success rate close to 100%; (2) rapid multiple genome editing using editing plasmids with different resistance markers; (3) using sacB, a counter-selection marker conferring sucrose sensitivity, to enable the active loss of the editing plasmids and facilitate multiple rounds of genetic modification or phenotypic analysis; (4) manipulation of essential genes by the creation of conditional mutants, using as example, polA encoding the DNA polymerase I essential for DNA replication and repair; (5) large DNA fragment deletion, up to 118 kb, from the Anabaena chromosome, corresponding to the largest bacterial chromosomal region removed with CRISPR systems so far. The genome editing vectors and the strategies developed here will expand our ability to study and engineer cyanobacteria, which are extensively used for fundamental studies, biotechnological applications including biofuel production, and synthetic biology research. The vectors developed here have a broad host range, and could be readily used for genetic modification in other microorganisms.
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  • 水产品种创制与高效养殖全国重点实验室
  • 中国科学院藻类生物学重点实验室
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  • 武汉白暨豚保护基金会
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  • 中国动物学会原生动物学分会
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  • 中国鱼类学会
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  • “一带一路”海域赤潮数据库
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  • 水生生物博物馆
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