Plant gRNA and Cas9 Coexpression Agrobacterium Binary Vector (Single gRNA)

The Agrobacterium binary vector system is a powerful and effective method for generating transgenic plants. This system utilizes the natural ability of the bacteria Agrobacterium tumefaciens to insert foreign DNA into the plant genome. To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system to a variety of plant species, VectorBuilder developed this guide RNA (gRNA) and Cas9 coexpression binary vector, enabling highly efficient generation of heritable targeted mutations in plants.

The Agrobacterium binary vector system is derived from the natural tumor-inducing (Ti) plasmid which contains a transfer DNA (T-DNA) region and a virulence (vir) region. The gene to be transferred is located in the T-DNA region between 25 bp direct repeat sequences, known as the left and right border repeats. In addition to the T-DNA region, the Ti plasmid contains the vir region which mediates the transfer of T-DNA and its integration in the plant genome. Our binary vector system was developed based on this mechanism with all tumor-associated intervening T-DNA sequences removed. The binary vector system achieves plant transformation using two vectors. The first, referred to as the T-DNA binary vector (or simply ‘binary vector’), contains the two T-DNA border repeats bracketing the DNA sequence which will be inserted into the plant host. The second vector is referred to as the vir helper plasmid. When the binary vector and the vir helper plasmid are both present in the same Agrobacterium cell through co-transformation, co-electroporation, or conjugation, proteins encoded by the vir helper plasmid mediate host genome integration of the sequence between the left and right border repeat elements.

The CRISPR-Cas9 system comprises a guide RNA (gRNA) and Cas9 protein, which together form a genome-editing complex. When a protospacer adjacent motif (PAM) is present on the non-targeted strand, the gRNA is able to bind to its complementary genomic sequence. The Cas9 nuclease then makes a double-strand break in the DNA followed by endogenous repair that typically results in mutations.

To achieve effective gene editing capability with the CRISPR/Cas9 system in plants, VectorBuilder has developed the gRNA and Cas9 coexpression binary vector. In this vector, the initiation and termination of gRNA transcription are respectively mediated by AtU6-26 (or OsU3) promoter and AtU6-26 terminator. Additionally, the Cas9 gene with maize codon-optimized sequence (ZmCas9) is driven by the CaMV 35S promoter and terminated by rbcS-E9 polyadenylation signal. This binary vector also carries a selectable marker such as Neo/Kana, Bar and Hygro. Like other binary vectors, all components to be transferred are delineated by left and right border T-DNA repeats. In addition to these segments to be transferred, this vector contains pBR322/pVS1 ori, permitting replication of the plasmid in Agrobacterium. Finally, the vector is equipped with the pVS1 StaA signal to increase the stability.

For further information about this vector system, please refer to the papers below.

Permanent integration of vector DNA:  Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of episomal DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transformation of plant cells with Agrobacterium vectors can deliver CRISPR components permanently into host plant cells due to the integration of the T-DNA region into the host genome.

Technical simplicity: Transformation of Agrobacterium with binary vectors is technically straightforward, as is transformation of plant cells using binary vectors and Agrobacterium.

Escherichia coli (E. coli) replication incompetency: This vector contains regions of replication that can only function in Agrobacterium.

3’ deletions: Within the plant, it is common for nucleolytic degradation to delete sequence from the T-DNA left boundary (e.g. 3’) end. However, this is generally not a significant concern since the user’s sequence of interest is cloned near the right boundary. However, degradation from the left boundary can affect the marker gene.

Integration of backbone sequences: In some cases, integration of vector backbone sequences may occur along with T-DNA boundary-flanked sequence. This phenomenon occurs less frequently when low copy Agrobacterium plasmids are used, such as in our binary vector system.

Promoter: The promoter driving your gene of interest is placed here.

gRNA: Guide RNA compatible with the Cas9 variant being used.

AtU6-26 terminator: Arabidopsis U6-26 gene terminator with downstream sequence. It allows transcription termination of small RNA transcribed by RNA polymerase III.

2×CaMV 35S: Double cauliflower mosaic virus 35S promoter. It is a strong plant ubiquitous promoter.

ZmCas9: Maize (Zea mays) codon-optimized CRISPR associated protein 9 from Streptococcus pyogenes with 3×FLAG tag and nuclear signal localization. It generates double-strand DNA breaks.

rbcsS-E9 polyA: Pisum sativum rbcS-E9 gene (encoding the small subunit of ribulose-1,5-bisphosphate carboxylase, rbcS). It allows transcription termination and polyadenylation of mRNA transcribed by RNA polymerase ll.

CaMV 35S_enhanced:  A strong chimeric promoter which drives marker expression.

Marker: A drug selection gene, allowing selection of plant cells transduced with the vector.

CaMV 35S pA: Cauliflower mosaic virus 35S polyadenylation signal. This facilitates transcription termination and polyadenylation of the marker gene.

LB T-DNA repeat: Left border repeat of T-DNA. Upon recognition by Ti plasmid in Agrobacterium, the region between the T-DNA border repeats is transferred to plant cells.

Kanamycin: Kanamycin resistance gene. It allows the plasmid to be maintained by kanamycin selection in bacterial hosts.

pBR322 ori: pBR322 origin of replication. It facilitates plasmid replication in E. coli. Plasmids carrying this origin exist in low copy numbers (15-20 per cell) in E. coli if Rop protein is present, or medium copy numbers (100-300 per cell) if Rop protein is absent.

pVS1 oriV: Origin of replication from the plasmid pVS1. It permits replication of low-copy plasmids in Agrobacterium.

pVS1 RepA: Replication protein from the plasmid pVS1. It permits replication of low-copy plasmids in Agrobacterium.

pVS1 StaA: Stability protein from the plasmid pVS1. It is essential for stable plasmid segregation in Agrobacterium.

RB T-DNA repeat: Right border repeat of T-DNA. Upon recognition by Ti plasmid in Agrobacterium, the region between the T-DNA border repeats is transferred to plant cells.