Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120
Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120
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DOI：
10.1021/acssynbio.8b00437
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发表时间：
2019-01-01
影响因子：
4.7
通讯作者：
Zhang, Cheng-Cai
中科院分区：
文献类型：
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作者：
Niu, Tian-Cai;Lin, Gui-Ming;Zhang, Cheng-Cai
CRISPR系统，如CRISPR- cas9和CRISPR- cpf1，已经成功地用于多种生物体的基因组编辑。虽然CRISPR-Cpf1技术最近已经在蓝藻中应用，但它的使用受到限制，没有充分利用这种强大的遗传系统的潜力。我们以蓝藻Anabaena PCC 7120为模型菌株，改进了工具，设计了基于CRISPR-Cpf1的遗传策略，使我们能够实现迄今为止难以在蓝藻中进行的遗传实验。这一进展包括：(1)单基因组修饰的“双间隔”策略，成功率接近100%；(2)利用具有不同抗性标记的编辑质粒进行快速多基因组编辑；(3)使用sacB（一种赋予蔗糖敏感性的反选择标记），使编辑质粒主动丢失，并促进多轮遗传修饰或表型分析；(4)通过创造条件突变体来操纵必要基因，例如，编码DNA复制和修复所必需的DNA聚合酶I的polA；(5) Anabaena染色体上的大片段DNA缺失，高达118 kb，这是迄今为止CRISPR系统所切除的最大的细菌染色体区域。这里开发的基因组编辑载体和策略将扩大我们研究和改造蓝藻的能力，蓝藻被广泛用于基础研究、生物技术应用（包括生物燃料生产）和合成生物学研究。本文开发的载体具有广泛的宿主范围，并且可以很容易地用于其他微生物的基因改造。
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.