A CRISPR/Cas9 toolkit for multiplex genome editing in plants

Abstract

Background: 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, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.

Results: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.

Conclusions: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Figure 1

Physical maps and structures of CRISPR/Cas9 binary vectors. (A) Physical maps of the backbones of pGreen and pCAMBIA from which CRISPR/Cas9 binary vectors were derived. The map of the helper plasmid required for propagation of pGreen in Agrobacterium and the mutated BsaI site on the pCAMBIA backbone are indicated. LB/RB, left/right border of T-DNA; pSa-ori, required for replication in Agrobacterium engineered with the corresponding replication protein (pSa-repA); KmR, kanamycin resistance gene; pUC-ori, replication origin required for replication in E. coli; pVS1-staA, pVS1-ori and pVS1-rep are the DNA elements required for replication in Agrobacterium. Only the 225-bp fragment between the LB and RB was left for comparison of the sizes of the pGreen and pCAMBIA backbones. (B, C) Physical maps of the regions between the RB and LB. The sizes of T-DNA regions and the structures of SpR-gRNA-Sc and final working gRNA are indicated. zCas9, Zea mays codon-optimized Cas9; U6-26p, Arabidopsis U6 gene promoter; U6-26t, U6-26 terminator with downstream sequence; OsU3p, rice U3 promoter; OsU3t, rice U3 terminator with downstream sequence; SpR, spectinomycin resistance gene; gRNA-Sc, gRNA scaffold.

Figure 2

Premade gRNA modules used for the assembly of two to four gRNA expression cassettes. (A) gRNA-expressing modules for both dicots and monocots. U6-29p, U6-26p, and U6-1p are three Arabidopsis U6 gene promoters; U6-29t, U6-26t, and U6-1t, corresponding Arabidopsis U6 gene terminators with downstream sequences; OsU3p and TaU3p, rice and wheat U3 promoters, respectively; OsU3t and TaU3t, rice and wheat U3 terminators with downstream sequences, respectively; gRNA-Sc, gRNA scaffold; DT1/2/3/4, dicot target-1/2/3/4; MT1/2/3/4, monocot target-1/2/3/4. The vector pCBC is the cloning vector into which the gRNA modules were inserted separately. (B) Examples of the assembly of two-gRNA expression cassettes for dicots and monocots using the gRNA modules. Note: Each PCR fragment is flanked by two BsaI sites (not shown).

Figure 3

Validation of maize codon-optimized Cas9 and three Pol-III promoters driving gRNA expression in maize protoplasts. (A) Sequence of the target site from the ZmHKT1 locus. The PAM, the putative cleavage site (red arrowhead), and the XcmI site (boxed) are indicated. (B,C) Mutation analysis by XcmI digestion of PCR fragments. GFP, 201, 301, 401 (B): PCR fragments amplified from the genomic DNA of maize protoplasts transfected with pUC-GFP (control), pBUN201-ZT1, pBUN301-ZT1, and pBUN401-ZT1, respectively. The three CRISPR/Cas9 vectors have the same gRNA but different Cas9: hCas9-1/2, two types of human-codon-optimized Cas9; zCas9, Zea mays codon-optimized Cas9. GFP, 401, 411, 421 (C): PCR fragments from the pUC-GFP, pBUN401-ZT1, pBUN411-ZT1, and pBUN421-ZT1 transfections, respectively; the three CRISPR/Cas9 vectors have the same zCas9 and gRNA, but the gRNA is driven by three different Pol-III promoters. − and + indicate whether the PCR fragments were digested with XcmI. Mutation efficiency (% indel) calculated based on the percent ratios of residual undigested PCR fragments (+ lanes: 569 bp) to total PCR products (− lanes); the WT indel values should be treated as the background level. (D,E) Alignment of sequences of mutated alleles identified from cloned PCR fragments resistant to XcmI digestion. The mutated alleles include deletions (D) and insertions (E). Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are shown. The type of indel and the number of indels of the same type are indicated.

Figure 4

Validation of the toolkit by targeted mutation of a maize gene. (A) Sequence of a region of maize ZmHKT1 with two target sites indicated. (B) Physical map of T-DNA carrying two-gRNA expression cassettes. The alignment of target of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (C) Mutation analysis of 20 T0 transgenic lines by XcmI or SphI digestion of PCR fragments. The lines used for sequencing analysis are indicated with boxes. (D) Alignment of sequences of mutated alleles identified from cloned PCR fragments from two representative T0 transgenic lines. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are displayed. The number of indels of the same type is indicated.

Figure 5

Validation of the CRISPR/Cas toolkit in Arabidopsis . (A) Physical maps of the T-DNAs of two pGreen-derived CRISPR/Cas9 binary vectors, each carrying two-gRNAs targeting three Arabidopsis genes (TRY, CPC and ETC2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Representative phenotypes of p2gR-TRI-A T1 transgenic lines. S, strong phenotypes similar to that of try cpc etc2 triple mutant, with highly clustered trichomes on leaf blades and petioles; M, moderate phenotypes with parts of leaf blades or a partial leaf blade displaying the phenotypes of the try cpc double mutant or the triple mutant; W, plants with weak or no mutant phenotypes. The total number of T1 transgenic plants, the number of T1 transgenic plants displaying strong, moderate, and weak phenotypes, and the percentage (in parentheses) of the total number are shown. The T0 seeds were screened on hygromycin MS plates for 13 days and grown in soil for 10 days before photographing. (C) Magnified image of a detached leaf displaying highly clustered trichomes on petioles, which is similar to the phenotype of the try cpc etc2 triple mutant. (D) Sequencing analysis of target gene mutations of a representative p2gR-TRI-A line. Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments. The type of indel and the number of indels of the same type are indicated.

Figure 6

The try cpc etc2 triple mutant can be differentiated from try cpc double mutant. Representative triple and double mutants and the wild type are shown. The seeds were sown on MS plates, vernalized at 4°C for 3 days, and transferred to an illumination incubator and allowed to grow for 10 days. The seedlings were transplanted to soil and allowed to grow for 17 days before photographing. The triple and double mutants were segregated from A17 T1 lines.

Figure 7

Validation of pCAMBIA-derived CRISPR/Cas binary vectors in Arabidopsis . (A) Physical map of T-DNA of the pCAMBIA-derived vector carrying two-gRNAs targeting two Arabidopsis genes (CHLI1 and CHLI2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Phenotypes of all transgenic seedlings from one screening. The T0 seeds were screened on hygromycin MS plates for 7 days, and all of the hygromycin-resistant seedlings were transferred to a fresh MS plate before photographing. The albino seedlings were numbered.