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About the Authors:
WenZhi Jiang
Affiliation: Department of Biochemistry, University of Nebraska, Lincoln, Nebraska, United States of America
Bing Yang
Affiliation: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa, United States of America
Donald P. Weeks
* E-mail:[email protected]
Affiliation: Department of Biochemistry, University of Nebraska, Lincoln, Nebraska, United States of America
Introduction
In recent years, zinc finger nuclease (ZFN) technology [1] and TAL Effector Nuclease (TALEN) technology [2]–[5] have become powerful gene editing tools for targeted gene modification in human cells, fruit flies, zebrafish, nematodes and plants. For both ZFNs and TALENs, engineered sequence-specific DNA binding domains are fused with a subunit of the nonspecific DNA nuclease, Fok1. As a result, pairs of ZFNs and TALENs targeting adjacent DNA target sites generate double strand breaks (DSBs) at or near the target site. Repair of the DSB by error-prone nonhomologous end joining (NHEJ) or homologous recombination (HR) often lead to gene sequence modification, including gene knockout. Within the past year, another highly promising system for gene editing, the clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system, has evolved from studies of bacterial defense systems that provide protection against invading viruses or plasmid DNAs [6]–[8]. CRISPR loci are variable short spacers separated by short repeats and are transcribed and processed into short non-coding RNAs. These short RNAs can form a functional complex with Cas proteins and guide the complex to cleave complementary foreign DNAs. The type II CRISPR/Cas system derived from Streptococcus pyogenes is the most widely used for gene editing [6]–[9]. It has the marked advantage of possessing a required PAM recognition sequence of only two nucleotides (GG). Development of single guide RNAs (sgRNAs) that are fusions of essential portions of tracrRNA with the “guide RNA” of crRNAs was an important improvement in facilitating rapid adoption of the CRISPR technology for targeted gene modification in eukaryotic cells [6],[9]. To obtain a functional RNA-guided gene disruption in a host cell, one needs only to transform the cell with the Cas9 gene and a gene (generally driven by an RNA polymerase III-dependent promoter) encoding a sgRNA that contains a 20 bp sequence complementary to the segment of DNA in the host cell that is the target for disruption by a DSB. Once...