Research Associate Professor Liu Jia from SIAIS published a review article in Cold Spring Harbor Perspectives in Biology, entitled Genome-Editing Technologies: Principles and Applications.
This article summarizes four classes of nucleases for gene-editing applications, including homing nuclease, zinc finger nuclease (ZFN), TALEN and CRISPR/Cas9. These nucleases can be customized to recognize and cleave designated genomic sequence, leading to double-stranded break, thereby facilitating gene knockout, gene deletion, transgene integration and nucleotide substitution. Homing nuclease can recognize 14-40 bp sequence with high specificity, however the difficulty of customization limits its widespread applications. ZFN is the first chimeric nuclease that can be synthesized in a modular manner, comprising of a zinc finger DNA-binding domain and a DNA-cleaving nuclease domain. Likewise, TALEN consists of a TALE DNA-binding protein derived from plant pathogens and a DNA-cleaving domain. TALEN have been widely used due to the facile assembly and high success rate compared to ZFN. Targeted DNA cleavage by CRISPR/Cas9 was first reported in 2012 and the ease with which it can be synthesized and manipulated has soon rendered it the most widely used gene-editing tool. In addition to the principles of the “molecular scissors”, this review summarizes a wide range of gene-editing applications using targeted nucleases, including construction of cell lines, creation of model animals, discovery of drug targets and treatment of human diseases, and also discusses future directions of gene-editing technologies.
Besides, this review article discusses the delivery methods of customized nucleases. Albeit the high gene-editing efficiency, the safe and efficient delivery of nucleases into targeted cells, particularly under complex environment suchin vivoconditions, remains one of the most important topics in the gene-editing field. During the graduate study, Dr. Thomas Gaj demonstrated for the first time that purified ZFN proteins can mediate gene editing in mammalian genome, and Dr. Liu Jia extended this protein-based gene-editing approach to TALEN as well as CRISPR/Cas9. Compared with traditional lipofection, nucleofection or viral transduction, purified nuclease proteins can induce gene modifications with higher efficiency and lower “off-target” effects, and thereby become widely used in recent gene-editing applications.
Dr. Thomas Gaj from University of California, Berkely is the first and co-corresponding author. Dr. Shannon S. Sirk from Stanford University is the second author. Graduate student Sai-lan Shui from ShanghaiTech University is the third author. Research Associate Professor Liu Jia is the co-corresponding author. This review was invited and edited by Professors Hamilton O. Smith, Clyde A. Hutchison III, J. Craig Venter and Daniel G. Gibson.
The working mechanisms of targeted nucleases