Development of the TECCDNA method

First, we developed the TECCDNA-based genome-editing method by working with the three homoeologues of TaGASR7 (TaGASR7-A1, -B1 and -D1), which have been implicated in the control of grain length and weight7. Each of the three homoeologues has three exons and two introns (Fig. 2a), and we designed 5 single guide RNAs (sgRNAs) that target exon 3 because it is highly conserved. After initial testing of nuclease activity in protoplasts8, the most effective sgRNA expression cassette (Supplementary Fig. 1 and Supplementary Table 1) was combined with that of Cas9 in a single DNA construct (pGE-TaGASR7, Fig. 2c). This was introduced by particle bombardment into immature embryos of two bread wheat varieties (Bobwhite and Kenong199). Embryogenic calli developed in 2 weeks, and a large number of seedlings (2–3 cm high) were regenerated in another 4–6 weeks. In contrast with most plant genome-editing experiments, we did not add herbicide or antibiotics to the medium to select for transformed plants (Fig. 1). Under our selection-free conditions, the total time from particle bombardment to obtaining testable plants was 6–8 weeks, which is 2–4 weeks shorter than the genome-editing protocol reported previously9.

Figure 2: Development and validation of the TECCDNA method. (a) Sequence of an sgRNA designed to target a site within a conserved region of exon 3 of TaGASR7 homoeologues. The protospacer-adjacent motif (PAM) sequence is highlighted in red and the BcnI restriction site is underlined. The outcome of PCR-RE assays analysing 12 representative tagasr7 mutants is shown. Lanes T0-1 to T0-12 show blots of PCR fragments amplified from independent wheat plants digested with BcnI. Lanes labelled WT/D and WT/U are PCR fragments amplified from WT plants with and without BcnI digestion, respectively. The bands marked by red arrowheads are caused by CRISPR/Cas9-induced mutations. (b) Genotypes of 12 representative mutant plants identified by sequencing. (c) Schematic of the structure of the pGE-sgRNA vector and five primer sets used for detecting transgene-free mutants. sgRNA refers to sgRNAs targeting TaGASR7, TaDEP1, TaNAC2, TaPIN1, TaLOX2, TdGASR7 and TaGW2, respectively. (d) Outcome of tests for transgene-free mutants using five primer sets in 12 representative tagasr7 mutant plants. Lanes without bands identify transgene-free mutants. Lanes labelled WT and plasmid show the PCR fragments amplified from a WT plant and the pGE-TaGASR7 vector, respectively. Full size image

We analysed the sgRNA target site in the regenerated T0 seedlings using PCR restriction enzyme digestion assay (PCR-RE assay)8, first using a conserved primer set (Supplementary Table 2) that recognizes all three TaGASR7 homoeologues and then with three primer pairs specific for each homoeologue (Supplementary Table 2). The mutagenesis frequency was defined as the number of plants carrying the observed mutations over the total number of bombarded immature embryos. For example, from 1,600 bombarded immature embryos of Bobwhite, a total of 80 tagasr7 mutants (an efficiency of 5.0%) with indels in the targeted region and another set of 21 such mutants from 800 bombarded immature embryos (2.6%) of Kenong199 were identified (Table 1). Targeted mutations were observed in all three homoeologues (Fig. 2a,b). Among the 80 Bobwhite mutants we identified nearly all combinations of mutations involving three TaGASR7 homoeologues, including 51 mutants with targeted indels in all three genomes (Supplementary Table 3). Importantly, 8 of these 51 mutants had all six alleles simultaneously knocked out (Supplementary Table 3). These data suggest that our new experimental scheme, based on TECCDNA in callus cells, is highly efficient in generating targeted mutations of TaGASR7 in T0 populations.

Table 1 The mutagenesis frequencies of three methods for wheat genome editing in T0 generation. Full size table

Validation of the TECCDNA method

Next, we examined whether the TECCDNA method was generally applicable for other wheat genes. We targeted the wheat orthologues of rice DEP1, NAC2 and PIN1 and a wheat lipoxygenase gene (TaLOX2). In rice, DEP1 controls inflorescence architecture and affects panicle growth and grain yield10; NAC2 has been found to regulate shoot branching11, and PIN1 is required for auxin-dependent adventitious root emergence and tillering12. TaLOX2 is highly expressed during grain development and may affect the storability of wheat grains13. We developed CRISPR/Cas9 DNA constructs for each of the four genes (Fig. 2c, Supplementary Fig. 1 and Supplementary Table 1), and bombarded immature embryos of Kenong199. The regenerated seedlings were analysed for mutations with conserved and specific primer sets (Supplementary Table 2) for TaDEP1. For simplicity, we designed only conserved primers (Supplementary Table 2) to detect mutations in TaNAC2, TaPIN1 and TaLOX2, the latter of which exists as a single copy (TaLOX2-D1 in the D genome). Targeted mutations were easily identified in all four genes in the T0 seedlings using PCR-RE analysis (Supplementary Figs 2 and 3). The mutagenesis frequency varied from 1.0 to 9.5%, and we identified 34 talox2-dd homozygous mutants among 76 mutant plants (44.7%; Table 1).

Applicability of TECCDNA method in durum wheat

In addition to bread wheat, durum wheat is also an important crop widely used for pasta foods. Because GASR7 is highly conserved in tetraploid and hexaploid wheat, the new method was also introduced into two different durum wheat varieties for inducing mutations in TdGASR7-A1 and -B1. The mutagenesis frequencies in tetraploid wheat lines exceeded 1.0%, and homozygous mutants resulting from simultaneous editing of all four alleles were obtained in T0 seedlings (Table 1 and Supplementary Fig. 4). To our knowledge, successful genome editing has not been reported for tetraploid wheat before this work. Clearly, TECCDNA-based genome editing provides a reliable genome-engineering method for this important crop.

Detection of transgenes

Because the mutant seedlings were developed in the absence of herbicide selection, there is a high probability that the CRISPR/Cas9 DNA construct is not integrated in the genome. This was investigated by examining the presence of plasmid DNA in the T0 mutants using PCR. We designed primer sets (Supplementary Table 2) specific for five discrete regions in the CRISPR/Cas9 DNA construct, representing all major parts (Fig. 2c). On the basis of the PCR analysis, the CRISPR/Cas9 DNA construct was found to be absent in 43.8% (cv Bobwhite; Fig. 2d) and 61.9% (cv Kenong199) of the T0 mutants for TaGASR7 (Table 1). In addition, we conducted Southern blot analysis for three independent tagasr7 mutants, without (T0-3 and T0-10) or with (T0-12) CRISPR/Cas9 DNA as detected with the above PCR assay. As anticipated, no hybridization signals were found in T0-3 and T0-10, although hybridizing bands were detected in T0-12 (Supplementary Fig. 5). Therefore, we could obtain some homozygous tagasr7 mutants with no detectable transgenes in the T0 generation (Table 1 and Supplementary Table 3). For the other four genes, our PCR analysis showed that the frequencies of mutants without detectable transgenes were 53.8% (TaDEP1), 75.0% (TaNAC2), 62.5% (TaPIN1) and 86.8% (TaLOX2) (Table 1). Likewise, absence of CRISPR/Cas9 DNA integration was found in 54.5–58.3% of the T0 mutants of the two durum wheat varieties (Table 1). Homozygous mutants with no detectable transgenes were also identified in TaLOX2 and TdGASR7 (Table 1). Thus, plants with targeted mutations and lacking active transgenes can be efficiently obtained using our TECCDNA genome-editing method.

Developing and testing the TECCRNA method

Although the chance of obtaining mutants with no detectable transgenes using the above method was high, a sizable proportion of the mutants still carried CRISPR/Cas9 DNA. Moreover, there is a possibility that the degradation products of CRISPR/Cas9 DNA may become integrated in the genome, which is difficult to be detected using PCR. Therefore, the genome-editing protocol was further optimized by transiently expressing the IVTs of Cas9-coding sequence and sgRNA in wheat callus cells (Fig. 1). The TaGW2 gene, which acts as a negative regulator of kernel width and weight in bread wheat14, was used to test this transient expression of CRISPR/Cas9 RNA (abbreviated as TECCRNA)-based genome-editing method. The target site of the TaGW2 gene was located in a conserved region in exon 8 (Fig. 3a, Supplementary Fig. 1 and Supplementary Table 1). We delivered Cas9 and sgRNA IVTs into the immature embryos of Kenong199 by particle bombardment, and regenerated the plants without herbicide selection as above (Fig. 1). From 1,600 bombarded immature embryos, 17 T0 mutants (efficiency 1.1%) were identified, among which 6 mutants (35.3%) contained site-specific indels in all six TaGW2 alleles (Fig. 3a, Table 1 and Supplementary Table 2). These site-specific indels were confirmed by Sanger sequencing (Fig. 3b). To the best of our knowledge, this is the first report of producing genome-edited plants with CRISPR/Cas9 IVTs. It is well known that, under normal growth conditions, RNA molecules are unlikely to become integrated into nuclear DNA in plant cells. Therefore, we believe that the mutants produced with CRISPR/Cas9 IVTs are free of external nucleic acid integration. However, we noticed that the mutagenesis frequency of TECCRNA-based genome editing (1.1%) was somewhat lower than that obtained with TECCDNA (3.3%) or conventional DNA integration-based genome-editing methods (3.0%) in a side-by-side experiment (Table 1). The relatively low mutagenesis frequency of the TECCRNA method may be because RNA was less stable and could be easily degraded compared with DNA.

Figure 3: Development and testing of TECCRNA method. (a) Sites within a conserved region of exon 8 of wheat TaGW2 homoeologues targeted by CRISPR/Cas9 systems. The PAM sequence is highlighted in red, the XbaI restriction sites are underlined and the single-nucleotide polymorphism in the targeted sequences is highlighted in green. Outcome of PCR-RE assays analysing nine representative tagw2 mutants is shown. Lanes T0-1 to T0-9 show blots of PCR fragments amplified from independent wheat plants digested with XbaI. Lanes labelled WT/D and WT/U are PCR fragments amplified from WT plants with and without XbaI digestion, respectively. The bands marked by red arrowheads are caused by CRISPR/Cas9-induced mutations. (b) CRISPR/Cas9-induced mutant TaGW2 alleles identified by sequencing. (c) Comparison the mutagenesis frequencies for three homoeologues of TaGW2 of three genome-editing methods. Full size image

Comparative analysis of off-target effects

Off-target is a main concern in current genome-editing studies. Thus, we compared potential off-target effects among the three wheat genome-editing methods outlined above. We computationally predicted the genome-wide potential off-target sites for TaGW2-sgRNA using the CasOT tool in bread wheat15. Eight likely off-target sites with three to four nucleotide mismatches to the recognition site of TaGW2-sgRNA were identified (Supplementary Table 4). However, none of these eight sites was mutated among the 67 tagw2 mutants (24 by conventional DNA integration-based method, 26 by TECCDNA method and 17 by TECCRNA method) detected using PCR-RE assay. Similarly, we found in the bread wheat genome 24 potential off-target sites for TaGASR7-sgRNA with two to five nucleotide mismatches (Supplementary Table 4). Again, none of these 24 sites were disrupted in the 101 tagasr7 mutants (including 80 in Bobwhite background and 21 in Kenong199 using the TECCDNA method).

Next, we examined off-target effects among highly similar bread wheat homoeologues using TaGW2-A1, -B1 and -D1 as example. The TaGW2-sgRNA recognition sequence was strictly conserved in TaGW2-B1 and TaGW2-D1, but had one-nucleotide mismatch to the cognate target site in TaGW2-A1 (Fig. 3a). We found that off-target frequencies caused by this one-nucleotide mismatch in TaGW2-A1 were lower than on-target mutagenesis frequencies in TaGW2-B1 and -D1 using all three methods. The conventional DNA integration-based genome editing, TECCDNA and TECCRNA methods induced on-target mutations in TaGW2-B1 (2.6%, 3.0% and 1.1%, respectively), TaGW2-D1 (2.9%, 2.9% and 1.1%, respectively) and off-target mutations in TaGW2-A1 (2.0%, 2.3% and 0.4%, respectively; Fig. 3c). The observed off-target effects may not be surprising because the one-nucleotide mismatch was located at position 9 of the protospacer-adjacent motif-proximal region of sgRNA, and many previous studies have found that mismatch around this position frequently leads to off-target mutagenesis16,17,18.

Mutation transmission and phenotypic analysis

To investigate whether the mutations produced in this work can be transmitted to the next generation, representative transgene-free T0 tagasr7, tadep1 and talox2 mutants were self-pollinated, and their T1 progenies were analysed using PCR-RE. For homozygous mutations detected in T0 (including those with simultaneous editing of all six alleles), the transmission rates were 100%; for the majority of the heterozygous mutants, Mendelian segregation occurred (homozygote/heterozygote/wild type (WT): 1:2:1; Supplementary Table 5). As anticipated, all the T1 individuals were transgene-free (Supplementary Table 5).

The phenotypic effects of the mutations in TaGASR7 and TaDEP1 were assessed. Homozygous, stable, transgene-free T2 mutants with all six alleles modified (that is, aabbdd) were identified and compared with WT controls. For TaGASR7, aabbdd mutant plants with frameshift mutations in all six alleles had significantly elevated thousand kernel weight (TKW), irrespective of the varietal background (P<0.01; Fig. 4a). This finding is consistent with the suggestion that TaGASR7 is a negative regulator of grain weight in bread wheat19. In the case of TaDEP1, the aabbdd mutant plants with frameshift mutations in all six alleles exhibited an extremely dwarf phenotype (with a mean plant height of 36.7 cm) compared with their WT counterparts (mean plant height: 56.0 cm; P<0.01; Fig. 4b,c). These results demonstrate for the first time that TaDEP1 is an important regulator of wheat growth and development.