In vitro CRISPR/Cas9 system for efficient targeted DNA editing

Yunkun Liu#, Weixin Tao#, Shishi Wen, Zhengyuan Li, Anna Yang, Zixin Deng, and Yuhui Sun*

mBio 2015, 6(6):e01714-1715

Epub Date: 10 November 2015

DOI: 10.1128/mBio.01714-15


The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3'→5' exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement inediting accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete therkDandhomEgenes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters.

Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system

Hu Zeng, Shishi Wen, Wei Xu, Zhaoren He, Guifa Zhai, Yunkun Liu, Zixin Deng, and Yuhui Sun*

Applied Microbiology and Biotechnology 2015, 99(24):10575-10585

Epub Date: 29 August 2015

DOI: 10.1007/s00253-015-6931-4


The current diminishing returns in finding useful antibiotics and the occurrence of drug-resistant bacteria call for the need to find new antibiotics. Moreover, the whole genome sequencing revealed that the biosynthetic potential of Streptomyces, which has produced the highest numbers of approved and clinical-trial drugs, has been greatly underestimated. Considering the known gene editing tool kits were arduous and inefficient, novel and efficient gene editing system are desirable. Here, we developed an engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) combined with the counter selection system CodA(sm), the D314A mutant of cytosine deaminase, to rapidly and effectively edit Streptomyces genomes. In-frame deletion of the actinorhodin polyketide chain length factor gene actI-ORF2 was created in Streptomyces coelicolor M145 as an illustration. This CRISPR/Cas9-CodA(sm) combined system strikingly increased the frequency of unmarked mutants and shortened the time required to generate them. We foresee the system becoming a routine laboratory technique for genome editing to exploit the great biosynthetic potential of Streptomyces and perhaps for other medically and economically important actinomycetes.

Delineating the biosynthesis of gentamicin X2, the common precursor of the gentamicin C antibiotic complex

Chuan Huang#, Fanglu Huang#, Eileen Moison, Junhong Guo, Xinyun Jian, Xiaobo Duan, Zixin Deng, Peter F. Leadlay*, and Yuhui Sun*

Chemistry & Biology 2015, 22(2):251-261

Epub Date: 29 January 2015

DOI: 10.1016/j.chembiol.2014.12.012


Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-L-methionine(SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.


Elucidation and optimization of biosynthetic pathway of microbial drugs


Linquan Bai*, Xuming Mao, Yongquan Li, Haoxin Wang, Yuemao Shen, and Yuhui Sun

生物产业技术 2015, (6):44-48

Biotechnology & Business 2015, (6):44-48 (Chinese)

Published online: 15 November 2015

DOI: doi:10.3969/j.issn.1674-0319.2015.06.006



English abstract is not available.

DNA 克隆和组装技术研究进展

Progress in DNA cloning and assembly techniques


Yanrong Shi, and Yuhui Sun*

微生物学通报 2015, 42(11):2229-2237

Microbiology China 2015, 42(11):2229-2237 (Chinese)

Published online: 4 May 2015

DOI: 10.13344/j.microbiol.china.150103



DNA cloning and assembly techniques are essential tools for molecular biology research. With the recent advances in synthetic biology, efficient and fast assembly of large DNAincluding anumber of genes is becoming more and more important. Meanwhile, a variety of DNA assembly methods are also developed very quickly. In this paper, various DNA assembly methods based on a typical enzyme digestion and ligation, PCR, homologous recombination, single strand annealing and splicing are summarized for providing effective technique tools for the further development of synthetic biology.


Aminoglycoside gentamicin research: fundamental progress and new application prospects


Xinyun Jian, Zixin Deng, and Yuhui Sun*

生物工程学报 2015, 31(6):829-844

Chinese Journal of Biotechnology 2015, 31(6):829-844 (Chinese)

Published online: 27 February 2015

DOI: 10.13345/j.cjb.140635



As an important aminoglycoside antibiotic, gentamicin has been used clinically over decades. With the development in modernbiological technology, the mechanisms of gentamicin action and resistance, its biosynthesis and structural modification were studiedin great depth. Meanwhile, its emerging novel bioactivities and potential applications are also under extensive exploration. Here we summarize the latest progresses and prospects towards the future development of gentamicin for more efficient and effective uses.