CRISPR/Cas9 technology-based monocotyledon gene knockout vector and application thereof

Abstract

The invention discloses a CRISPR / Cas9 technology-based plant gene knockout vector and an application thereof. The plant gene knockout vector is monocotyledon CRISPR / Cas9 knockout vector pCAMBIA1300DM-OsU3(BspQ I)-Cas9, and contains a BspQ I insertion site. The CRISPR / Cas9 plant knockout binary vector pCAMBIA1300DM-OsU3(BspQ I)-Cas9 commonly constructed by an sgRNA-OsU3 sequence composed of the BspQ I insertion site and a 2x35s-hSpCas9-ter sequence can realize high-efficiency rapid construction of the monocotyledon gene knockout vector through a one-step restriction and ligation method; and the CRISPR / Cas9 knockout vector pCAMBIA1300DM-OsU3(BspQ I)-Cas9 has the characteristics of high targeting efficiency and low off-target efficiency, and can be applied to plant target gene knockout or applied to plant gene editing in a kit form.

Description

[0001] Technical field: [0002] 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 [0003] 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 c...

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

[0005] There is also a system that integrates the promoter, sgRNA scaffold and Cas9 fragments into a binary vector, and selects BsaI as the insertion site, but the defect of this system is that there is a SpR (spectinomycin resistance gene) between the two BsaIs. Mycin resistance gene) fragment, during the construction process, Bsa I enzyme digestion is required to recover the linearized vector, and then connect to the target site (HL Xing, L Dong et al., 2014)

Images