CRISPR tool shows new potential in gene editing

Accurate DNA / gene replacement is a promising genome editing tool and can be easily extended to molecular engineering and design breeding. Currently, CRISPR / Cas9 system can be well used as a tool for plant gene knockout. For instance, Chinese Academy of Agricultural Sciences CRISPR achieved soybean genome editing; Anhui Academy of Agricultural Sciences made use of CRISPR to edit rice genome; applying genome editing technology in plant genes functional identification and crop breeding. However, gene replacement in plants has been rarely reported.

A simple method for precisely targeted genome editing could have important applications in terms of functional identification and genetic improvement of plant genes. In recent years, meganuclease 1, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), has been developed as a sequence-specific nuclease, for targeted double-strand breaks (DSBs) introducing DNA, in order to let us conduct gene editing through endogenous DNA damage repair pathways.

Recently, researchers have associated nuclease based CRISPR system, developed a new guide RNA gene editing tools. CRISPR / Cas9 nuclease system, can be a chimeric RNA (a so-called single guide RNA, sgRNA) targeting a particular genomic locus, as compared with protein targeting tool guide, have a higher degree of flexibility targeting selection sex. CRISPR / Cas9 system has been proven to promote genome editing different species, including mammals (including humans), microorganisms and plants.

Use CRISPR / Cas9 achieve knockout, it represents the first application of this system, because the Cas9 induced DSBs, can be connected via a non-homologous end (NHEJ) mechanism to repair, which is an error-prone repair pathway, may be introduced in the DNA repair process short deletion or insertion. HDR (homology-directed repair), is an alternative method for repair of DSBs chromosome, the genome of the plant engineering more attractive, because it can achieve more subtle modifications of the DNA sequence, including DNA correction, replacement or targeted gene knock , or any type of mutation.

HDR-dependent target gene replacement or knock provides an unprecedented opportunity for genome editing, but also more challenging because the targeted DSB (s) must be a recovery template coexist. Using a pair of the sgRNAs Cas9 TALENs or nucleases can generate targeted genomic deletion mutation. Dual-induced DSB damage or deletion mutation, targeted gene replacement is also a necessary step.

Stable transformation CRISPR / Cas9 of, and a vector genome editing necessary element contains (by Agrobacterium-mediated transformation), by identifying "non-transgenic" organism easily screened. About HDR-mediated gene targeting, Fauser et al demonstrated that nuclease and nicking enzyme systems are effective tools. When paired sites near each other, the precise design paired sgRNA / Cas9 nicking enzyme neighbor - which may have enhanced specific method can be used for targeted gene insertion, gene stacking and Knock. However, to date, no study in vivo targeted gene replacement event created in plants, but also has a lower risk of GM contamination.

In this study, the researchers targeted two MIRs (AtMIR169a and AtMIR827a) two flanking region through a pair of passing sgRNAs. They first designed a double sgRNA / Cas9 carrier composition (construct # 1), successfully removed the miRNA gene regions --MIR169a and MIR827a. By PCR and subsequent sequencing, the researchers validated these shortcomings, so MIR169a and MIR827a sites generate 20% and 24% deleted efficiency.

Then, they achieved the targeting switch of a target gene through the stable conversion between CRSPR / Cas9 and a donor template DNA donator templater, in which the same point of the sgRNA target plant is designed to remove the target gene and remove DNA donator which promotes HDR. These findings demonstrate an alternative strategy which uses CRSPR / Cas9 system for genome editing by gene replacement.

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