AtGCS promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by Agrobacterium rhizogenes-mediated transformation

Abstract

Agrobacterium rhizogenes-mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive Cas9 expression. Here, we identified a highly efficient Arabidopsis thaliana gamma-glutamylcysteine synthetase promoter, termed AtGCSpro, with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. AtGCSpro achieved higher homozygous/biallelic mutation efficiency than the most widely used CaMV 35S promoter in driving Cas9 expression in soybean, Lotus japonicus, and tomato roots. Using the pAtGCSpro-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p2 × 35Spro-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the pAtGCSpro-Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing. AtGCSpro is conservatively active in various eudicots species, suggesting that AtGCSpro might be applied in a wide range of dicots species.

Keywords: CRISPR/Cas9; agrobacterium rhizogenes–mediated transformation (ARM); gamma-glutamylcysteine synthetase gene; genome editing; hairy root; homozygous/biallelic mutation.

Figure 1

The schematic diagrams of vectors. The schematic diagrams of the pRedGa1 (AtGCSpro ₂₄₁₁:: GUSPlus), pRedGa2 (AtGCSpro ₁₉₇₇:: GUSPlus), pRedGa3 (AtGCSpro ₁₆₂₉:: GUSPlus), pRedGa4 (AtGCSpro ₁₁₇₈:: GUSPlus), and pRedGa5 (AtGCSpro ₈₃₃:: GUSPlus) (A). CRISPR/Cas9-mediated gene knockout vector backbones pRd35Cas9, pRdGa1Cas9, pRdGa4Cas9, pRdGa5Cas9, pRdUbiCas9, pRdYCas9, and pMd35Cas9 (B).

Figure 2

Histochemical localization of GUS activity in the pAtGCSpro ₂₄₁₁:: GUSPlus hairy roots in broad dicots species by ARM transformation. High level of GUS activity was accumulated in the whole transformed pAtGCSpro ₂₄₁₁:: GUSPlus roots in soybean (A, B), tomato (C, D), cucumber (E, F), L. japonicas (G), cotton (H), tobacco (I, K), sweet potato (J, L), respectively. White arrows indicate the transgenic roots. Pictures B, D, F, K, L are closed up of sections A, C, E, I, J marked in the orange boxes, respectively. The GUS signal was found in the teratoma where hairy roots can emerge (indicated by orange arrows). All composite plants were observed from 16 to 21 d post-infected seedlings with K599 carrying pRedGa1 construct. Bars = 5 mm.

Figure 3

AtGCSpro activity assay with different shortened lengths in soybean hairy roots by ARM transformation. AtGCSpro ₁₉₇₇:: GUSPlus (A, B). AtGCSpro ₁₆₂₉:: GUSPlus (C, D). AtGCSpro ₁₁₇₈:: GUSPlus (E, F). AtGCSpro ₈₃₃:: GUSPlus in the soybean hairy roots (G, H). The transgenic root marked in the black box in picture (G) is closed-up (I). White arrows indicate the transgenic roots. Pictures B, D, F, and H are closed-up of sections (A, C, E), and G marked in the orange boxes, respectively. The GUS signal was found in the teratoma where hairy roots can initiate (indicated by orange arrows). All composite plants were observed from 16-day-old post-infected seedlings. Bars = 1 cm.

Figure 4

AtGCSpro ₁₁₇₈ activity assay in other dicots hairy roots by ARM transformation. Cucumber (A) and tomato (B) transformed with AtGCSpro ₁₁₇₈:: GUSPlus. GUS signal can be observed in the region where will produce transgenic roots (Shown by orange arrows in (C and D) (C, D). Pictures C and D are closed-up of sections (A, B) marked in the orange boxes, respectively. All composite plants were observed from 16-day-old post-infected seedlings with K599 carrying pAtGCSpro ₁₁₇₈:: GUSPlus. Bars = 1 cm.

Figure 5

Identification of CRISPR/Cas9-induced mutation in the GmNARK (Rj7) target loci in soybean with AtGCSpro ₂₄₁₁ and 2×35S to drive Cas9, respectively. The sequence of an sgRNA designed to target a site within the first exon region of Rj7. The protospacer-adjacent motif (PAM) sequence is highlighted in blue and the EcoRI restriction site is underlined (A). PCR-RE assays to detect CRISPR/Cas9-induced mutation in the Rj7 target loci from 30 different independent p2×35Spro-Cas9 hairy roots (B). Genotypes of eight representative mutants from transformed with p2×35Spro-Cas9 hairy roots identified by sequencing (C). PCR-RE assays to detect CRISPR/Cas9-induced mutation in the Rj7 target loci from 30 different independent pAtGCSpro ₂₄₁₁-Cas9 hairy roots (D). Genotypes of six representative rj7 mutants from transformed with pAtGCSpro ₂₄₁₁-Cas9 hairy roots identified by sequencing (E). In sections B and D, Lanes WT and WTE, undigested PCR amplification fragment and digested wild-type controls by EcoRI, respectively. Lanes 1–30, different independent transgenic hairy roots. In sections C and E, deletions and insertions are indicated as dashes and blue letter, respectively. The types and number(s) of indels are indicated in the right column. Examples given of mutation at target site in the p2×35Spro-Cas9 and pAtGCSpro ₂₄₁₁-Cas9 hairy root, respectively (F). Black arrows indicate the site of indels mutation. The PAM regions and mutated target sites are shown in the black box.

Figure 6

PCR-RE assay mutation efficiency in the NRSYM1/LjNLP4 target loci in L. japonicus. Sequence of an sgRNA designed to target a site within the first exon region of LjNLP4. The PAM sequence is highlighted in blue and the BamHI restriction site is underlined (A). L. japonicus hairy roots with edited NRSYM1/LjNLP4 allele. Transgenic hairy roots with edited LjNLP4Ljnlp4 (white arrow indicated), Ljnlp4Ljnlp4 allele (red arrow indicated) in picture a, and the wild type in picture e; pictures b, c, and d are closed-up of sections a (big white box), a (small white box) and e marked in the boxes, respectively. Bars = 1 mm (B). PCR-RE assays to detect mutation efficiency in the NRSYM1/LjNLP4 target loci. Lanes 1–16, different independent transgenic hairy roots. Lanes WT and WTB, undigested PCR amplification fragment and digested wild-type controls by BamHI, respectively (C, D). Five lines (#4, #6, #10, #14, and #15) were homozygous or biallelic mutations (C). 13 lines (#1, #3-7, #9-13, #15, and #16) were homozygous or biallelic mutations (D). An example shown of mutation in the target site in p2×35Spro-Cas9 and pAtGCSpro ₁₁₇₈-Cas9 hairy root, respectively (E).