Monocotyledon plant gene knockout vector based on CRISPR/Cas9 technology, and applications thereof

Abstract

The invention discloses a plant gene knockout vector based on a CRISPR / Cas9 technology. The monocotyledon plant gene knockout vector is characterized in that the plant gene knockout vector is a monocotyledon plant CRISPR / Cas9 knockout vector pCambin1300-OsU3-Cas9, and contains the AarI insertion site. According to the present invention, with the CRISPR / Cas9 plant knockout double-component vector pCambin1300-OsU3-2x35s-Cas9 formed from a sgRNA-OsU3 structure sequence comprising the AarI insertion site and a 2x35s-Cas9-ter sequence, the purpose of the efficient and rapid construction of the monocotyledon plant gene knockout vector can be achieved through the one-step digestion linkage method; and the provided CRISPR / Cas9 knockout vector pCambin1300-OsU3-2x35s-Cas9 has characteristics of high targeting efficiency and extremely low undershoot efficiency, and can be used for plant target gene knockout or can be used for plant gene editing in a kit form.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a monocot gene knockout vector based on CRISPR / Cas9 technology and an application thereof. Background technique [0002] Type II CRISPR / Cas9 system is an acquired immune system from Streptococcus, which can resist the invasion of foreign genes. After artificial modification, this system has been widely used in animals and plants. It is mainly composed of CRISPR ( C lustered R regularly I Interspaced S hort P alindromic R epeats) and specific Cas9 protein composition. CRISPR is a cluster of regularly interspaced short palindromic DNA repeats, which consists of a series of short highly conserved direct repeats and sequence intervals of similar length. Cas9 protein is a multi-domain protein consisting of 1409 amino acids, containing two nuclease domains RuvC-like and HNH. The CRISPR / Cas9 system was first successfully applied to genome editing in eukaryotic cells by...

AI Technical Summary

Problems solved by technology

In terms of the commonly used Agrobacterium plant transformation, the construction process of the plant gene knockout vector system based on CRISPR / Cas9 technology is relatively cumbersome, requiring multiple steps to integrate each part into the binary vector

In some reported CRISPR / Cas9 systems, the sgRNA is first inserted into the pATU6-26SK / pOsU6SK vector with the help of the restriction endonuclease BbsI, and then the AtU6-sgRNA fragment is obtained by restriction endonuclease KpnI and SalI or KpnI The OsU6-sgRNA fragment was obtained by double digestion with HindIII, and at the same time the digested SalI-Cas9-EcoRI or HindIII-Cas9-EcoRI fragment was connected with the linearized plasmid pCAMBIA1300 digested with KpnI and EcoRI to construct a binary vector. The construction process of this system is relatively complicated and the cycle is long (ZY Feng, JK Zhu et al., 2013); in addition, there is a synthetic method to directly synthesize the promoter and sgRNA unit, and then construct it on the binary vector. High, long period (Wenzhi Jiang et al.,2013)

In terms of gene gun transformation, the reported systems are to co-wrap the pU6-gRNA or pU3-gRNA vector and the Cas9 vector into the plant tissue, which is relatively expensive.

For the above systems, to complete the construction of a plant gene CRISPR / Cas9 knockout vector, either the cost is high or the process is cumbersome, and it cannot meet the needs of high-throughput plant gene CRISPR / Cas9 knockout vector construction

[0004] There is also a system that integrates the promoter, sgRNA scaffold and Cas9 fragments into a binary vector, and selects Bsa I as the insertion site, but the defect of this system is that there is SpR (spectinomycin resistance) between the two Bsa Is. gene spectinomycin resistance gene) fragment, during the construction process, it needs to be digested with Bsa I to recover the linearized vector, and then connected to the target sites (target sites) (HL Xing, L Dong et al., 2014)

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