Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice

Abstract

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

Figure 1.

Mechanism for targeted gene disruption by the Cas9/sgRNA complex and subsequent mutagenesis by NHEJ DNA repair. Before targeted DNA cleavage, Cas9 stimulates DNA strand separation and allows a sgRNA to hybridize with a specific 20 nt sequence in the targeted gene (in this case, a non-functional, mutant GFP gene). This positions the target DNA into the active site of Cas9 in proper orientation in relation to a PAM (tandem guanosine nucleotides) binding site. This positioning allows separate nuclease domains of Cas9 to independently cleave each strand of the target DNA sequence at a point 3-nt upstream of the PAM site. The double-strand break then undergoes error-prone NHEJ DNA repair during which deletions or insertions of a few nucleotides often occurs. Those approximately one in three deletions or insertions that restore a proper reading frame in the gene’s coding region allow for restoration of gene activity.

Figure 2.

Strategy for use of the Cas9/sgRNA system for mutagenesis and restoration of activity of a non-fuctional mutant GFP gene following Agrobacterium-mediated delivery of the Cas9 and sgRNA genes to Arabidopsis or tobacco cells. The Cas9 and sgRNA genes reside on a single binary vector that is carried by one A. tumefaciens line and the mutant GFP gene is present on a different binary vector in a separate A. tumefaciens line. Following infiltration of leaves with a mixture of the two A. tumefaciens lines and co-transformation of single cells, a specific 20-nt DNA sequence in the mutant GFP gene is targeted by the Cas9/sgRNA complex for cleavage. Repair of the double-strand DNA break by NHEJ results in deletion or insertion of nucleotides at the cleavage site and, in some case, restoration of a functional GFP gene, the product of which can be observed by fluorescence confocal microscopy.

Figure 3.

Red chlorophyll fluorescence signals and GFP signals from Arabidopsis leaf cells. (A) Leaf cells infiltrated with A. tumefaciens carrying a vector containing a wild-type GFP gene. (B) Leaf cells infiltrated with a mixture of two A. tumefaciens lines, one with a vector containing a non-functional mutant GFP gene and the other line carrying a vector for the expression of the Cas9 gene and the sgRNA gene. Expression of GFP in these leaf cells can occur only if a DNA target site in the non-functional GFP is recognized and cleaved by a Cas9/sgRNA complex, and resulting error-prone DNA repair by NHEJ leads to deletion or insertion of nucleotides that restore a proper reading frame in the GFP gene-coding region. Leaves were photographed at 40× and 600× magnification 48 h after A. tumefaciens infiltration.

Figure 4.

Red chlorophyll fluorescence signals and GFP signals from tobacco leaf cells. (A and C) Two examples of tobacco leaf cells infiltrated with a mixture of two A. tumefaciens lines, one with a vector containing a nonfunctional, mutant GFP gene and the other line carrying a vector for the expression of the Cas9 gene and the sgRNA gene. Green fluorescence is due to restoration of a proper reading frame of the GFP gene due to Cas9/sgRNA-mediated targeted gene cleavage and subsequent error-prone NHEJ DNA repair. (B and D) The images displayed in (A) and (C) merged with images of red fluorescence from chlorophyll. Leaves were photographed at 600× magnification 48 h after A. tumefaciens infiltration.

Figure 5.

DNA sequences of initially non-functional GFP genes following target site cleavage by Cas9/sgRNA and mutagenesis by NHEJ DNA repair in transgenic Arabidopsis and tobacco leaf cells expressing Cas9 and sgRNA genes. The DNA sequence of the starting non-functional mutant GFP gene is provided above each set of Cas9/sgRNA-mutagenized gene sequences derived from Arabidopsis (top set) and tobacco (bottom set). The 20 nt target sequence for the Cas9/sgRNA complex is in blue, the PAM site in red, and the ApaLI recognition site is underlined in blue. For the Cas9/sgRNA-mutagenized DNA sequences, deleted nucleotides are depicted as red dots and inserted nucleotides are shown in green. The net length of insertions and/or deleletions (In/Del) and the frequency with which each DNA sequence pattern was observed (Freq.) are presented in the columns to the right. One sequence in the Arabidopsis set and one sequence in the tobacco set arose from sequential deletions and insertions (first and second lines of each DNA sequence set, respectively).

Figure 6.

Gene constructs in a binary vector used in transformation of immature sorghum embryos to test for Cas9/sgRNA activity. A single binary vector was designed and constructed to contain (A) a chimera of a wild-type GFP gene and a neomycin phosotransferase gene, (B) a non-functional mutant DsRED2 gene, (C) a Cas9 gene codon-optimized for expression in maize, (D) a sgRNA gene driven by a rice U6 promoter and targeting the mutant DsRED2 gene.

Figure 7.

Clover (green) fluorescence protein signals and DsRED2 fluorescence protein signals from immature sorghum embryo cells. An immature sorghum embryo was co-cultivated with A. tumefaciens cells carrying a binary vector containing four genes driven by plant gene promoters (i.e. a clover fluorescence gene, a mutant non-functional DsRED2 gene containing a 20-bp CAs9/sgRNA target site, a Cas9 gene and a sgRNA gene driven by a rice U6 gene promoter) as described in Figure 6. (A) Image of cells of the immature embryo expressing the clover fluorescence protein. (B) Image of cells expressing red fluorescence protein produced from a mutagenized nonfunctional DsRED2 gene converted by the action of a Cas9/sgRNA complex and subsequent NHEJ into a functional DsRED2 gene. (C) Merged image of A and B confirming expression of clover fluorescence protein in a cluster of adjacent immature embryo cells and co-expression of clover fluorescence protein and DsRED2 fluorescent protein in an immediately adjacent cluster of transformed cells. Embryo cells were photographed 2 weeks following co-cultivation with A. tumefaciens carrying the Y158 binary vector.

Figure 8.

Gene constructs used in transformation of rice protoplasts to test for Cas9/sgRNA activity and test results. (A) The S.pyogenes Cas9 gene driven by the CaMV 35S promoter and containing a Nos gene termination region. (B) A synthetic single guideRNA gene targeting the promoter region of the rice OsSWEET14 gene and containing a hexathymidine U6 gene termination signal. (C) Diagram showing the 20-nt sequence of the OS11N3 gene (black) that is the target for hybridization to 20 nt in the synthetic sgRNA molecule (red). The SexAI restriction enzyme recognition site is underlined. (D) Diagram showing the DNA sequence of the target region of a OsSWEET14 gene (red) in which Cas9/sgRNA-mediated DNA cleavage and subsequent error-prone NHEJ DNA repair have caused mutagenesis leading to deletion of 9 nt (red dashes) from a critical region of the OsSWEET14 gene promoter. SexAI DNA recognition sequence is underlined in the wild-type sequence. (E) Diagram showing the DNA sequence of the target region ofOsSWEET11 gene in which Cas9/sgRNA-mediated DNA cleavage and subsequent error-prone NHEJ DNA repair have caused mutagenesis leading to deletion (dashes) and substitutions (red letters) at the expected sgRNA target site in the OsSWEET11 gene promoter. The DNA sequence recognized by BsrGI is underlined in the wild-type sequence.