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BIT Has Achieved Important Research Results in the Optimization and Application of Prokaryotic Genome Editing Technology

release date :2019-09-27 08:39:00  |   [ close window ]ViewCount:

     Translator: News Agency of BIT, Pang Yu

    Editor: News Agency of BIT

Recently, Prof. Huo Yixin’s research group of School of Life Science, BIT, has once again achieved important research results in the optimization and application of prokaryotic CRISPR genome editing technology, and published in the TOP Journal Applied Microbiology and Biotechnology. The first author of the thesis is Huang Chaoyong, a 2019 doctoral student and the corresponding author of the communication is Prof. Huo. Zhang Xueli and Bi Changhao, researchers of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and Ma Xiaoyan, a researcher of BIT participated in the research, which is funded by National Natural Resources Fund project, Ministry of Science and Technology key research and development projects, BIT science and technology innovation project, and Suzhou Industrial Park. Part of the research work was completed in the Joint Laboratory of BIT-SZ-UCLA-ITA. The research group has just published research results in related fields in the Q1 Journal Microbial Cell Factories. The first author of the thesis is Zhang Jiao, a 2019 master graduate, the corresponding author of the communication is Prof. Huo, and Jiang Yu, the researcher of CAC Center for Excellence in Molecular Plant Sciences/Institute of Plant Physiology and Ecology also participated in the research.

As a new generation of genome editing technology, CRISPR/Cas9 has been widely used in genome editing of animals, plants and microorganisms. This technique utilizes SpCas9 protein from Streptococcus pyogenes to generate site-specific DNA double-strand breaks, which are repaired by homologous recombination (HR) or non-homologous end-connection (NHEJ) mechanisms, and introduce the mutations needed in the genome. Unlike eukaryotes, prokaryotes are generally difficult to repair DNA double-strand breaks. Most bacteria lack the NHEJ system and only have inefficient HR systems. Therefore, most researchers choose to combine CRISPR/Cas9 with recombination engineering to help prokaryotic organisms repair DNA double strand breaks by over-expressing the bacteriophage-derived λ-Red system (a highly efficient HR system), thereby improving editing efficiency. Compared with NHEJ-mediated genome editing, HR-mediated genome editing requires a donor DNA as an editing template, which complicates genome editing operations. Studies have shown that some prokaryotes have simplified versions of NHEJ systems similar to eukaryotes, which require only two functional proteins: Ku and LigD. Escherichia coli has long been considered to lack NHEJ system. In recent years, domestic researchers have introduced NHEJ system derived from Mycobacterium tuberculosis and M. smegmatis into Escherichia coli, and successfully developed an HR-independent genome editing method. It is used to quickly delete the E. coli genome sequence, which greatly accelerates the process of genetic transformation of E. coli. In fact, researchers abroad have proved that E. coli has a terminal attachment system similar to NHEJ (the researchers named it A-EJ). Its functional proteins are not Ku and LigD, but RecBCD and LigA. Since A-EJ is far less efficient than NHEJ in repairing DNA double strand breaks, there are no reports of genome editing using A-EJ. Is it possible to replace NHEJ with A-EJ for genome editing? The answer is yes.

Fig. 1. CNEE-based genome editing process

Prof. Huo Yixin’s research group of School of Life Science, BIT, constructed a double-plasma rigorously inducible genome using xCas9-3.7 (a variant of SpCas9) developed by David Liu’s research group and a sgRNA (an enhanced version of tracrRNA: crRNA) engineered by the Jennifer Doudna’s research group, and named it CNEE. The system is extremely simple, with only the functional components of Cas9 and sgRNA, without any exogenous DNA repair proteins. Using this system, genome editing can be carried out efficiently even if the efficiency of the host DNA repair system is very low. With the help of host A-EJ system, CNEE system was introduced into Escherichia coli, and efficient gene knockout and deletion of large fragments up to 83 KB were achieved (Fig. 1). This method does not require editing templates, and can achieve rapid iteration of E. coli genetic modification, and editing efficiency does not depend on high transformation activity of host cells. Since RecBCD and LigA (or their homologous genes) are present in all prokaryotes, the CNEE system is theoretically applicable to genome editing of any prokaryote. Subsequently, a mutation system on the basis of CNEE was constructed. For the convenience of description, the system is temporarily called CNEE-V1. The introduction of CNEE-V1 into E. coli enables efficient and accurate genome editing, including deletion, insertion and substitution of sequences, with the assistance of the host RecA homologous recombination system. CNEE-V1 is an effective supplement to CNEE. Since RecA (or its homologous gene) is present in all prokaryotes, CNEE-V1 is also theoretically applicable to genome editing of any prokaryote. In addition, the research group also proved that the introduction of mycobacterial-derived NHEJ system into CNEE can further enhance the performance of CNEE. Similarly, the performance of CNEE-V1 can be further enhanced by incorporating the phage-derived λ-Red system. Related research results are published in the TOP Journal Applied Microbiology and Biotechnology with the link of the article: https://doi.org/10.1007/s00253-019-10104-w

Fig. 2. Genetic modification of Corynebacterium glutamicum to increase isobutyric acid production

CRISPR/Cpf1 is a genome editing system developed after CRISPR/Cas9. Compared with CRISPR/Cas9, CRISPR/Cpf1 has a lower off-target rate and requires less nuclease and RNA molecules, and thus has great application potential in the field of genome editing. However, the relatively low editing efficiency limits the application of the CRISPR/Cpf1 gene editing system. Corynebacterium glutamicum is an important industrial strain. Previously, the efficiency of genome editing in Corynebacterium glutamicum using CRISPR/Cpf1 was less than 15%, and linear DNA could not be used as a repair template. Therefore, the research team systematically optimized the CRISPR/Cpf1 system for genome editing of Corynebacterium glutamicum, and optimized parameters including PAM sequence, length of spacer sequence, and type of repair template. The efficiency of genome editing in Corynebacterium glutamicum using the optimized CRISPR/Cpf1 system is significantly improved, and linear DNA can be used as a repair template. Subsequently, the optimized CRISPR/Cpf1 system was used to genetically modify Corynebacterium glutamicum. The yield of isobutyric acid was successfully increased by knocking out the genes pyc, ldh and adhA (Fig. 2). Related research results are published in Microbial Cell Factories with the link of the article: https://doi.org/10.1186/s12934-019-1109-x

 

Profiles of Prof. Huo Yixin:

Prof. Huo Yixin graduated from Nankai University and got his doctor’s degrees in Peking University and Université Paris Diderot-Paris 7. He has studied and worked for 13 years at Pasteur Institute in France, University of California, Los Angeles and other European and American research institutes and biotechnology companies, and he has also been engaged in basic research and applied transformation research in the academic and industrial fields. Since 2016, Prof. Huo has set up a metabolic engineering research group at BIT, focusing on “microbial refining and manufacturing of natural resources”, considering “design-construction-screening-amplification of artificial cell factories” as the main line, and taking a variety of model organisms as the research object, a series of studies have been carried out, and a number of research results have been achieved. Since 2018, as the corresponding author or the first author, Prof. Huo has published articles in Nature Communications (1), Metabolic Engineering (1), Applied Microbiology and Biotechnology (4), Current Opinion in Biotechnology (1), ACS Synthetic Biology (1), Microbial Cell Factories (2), Engineering (1), Journal of Biotechnology (1) and JoVE (1). Moreover, he has also applied for multiple invention patents.

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