For 10,000 years, humans have utilized the genetic diversity generated from spontaneous mutations and recombination for the selection of improved crops. These traditional breeding approaches have been extremely successful in delivering elite crop varieties with high yields and other enhanced traits, and even today, they remain the corner stone of plant breeding. In recent times, these classical breeding approaches could be accelerated by increasing selection efficiency using marker-assisted selection [1] and genomic selection [2]. However, the more knowledge we obtain about the underlying genomic factors of yield and quality, the more the limitations of these traditional breeding approaches become apparent. Due to the random nature of recombination and undirected mutagenesis, further improvement of current elite germplasm is a lengthy and tedious process. Introgression of beneficial traits into an elite variety is often impaired by linkage drag, the transfer of deleterious genetic material genetically linked to the desirable trait. This often necessitates multiple rounds of backcrossing and selection to restore the elite background, which is highly time- and cost intensive [3]. Furthermore, the efficiency of classical breeding approaches depends on the amount of available functional diversity, which is limited in many elite varieties that have passed through genetic bottlenecks during domestication [4]. Thus, the reliance on natural or randomly induced diversity is a limiting factor slowing down the breeding process [5] and contributing to an unpredictable breeding outcome [6]. In contrast, the highly precise nature of the genome editing technology CRISPR/Cas enables an unparalleled level of control over the mutation process, allowing immediate pyramiding of multiple beneficial traits into an elite background within one generation [7]. Additionally, direct improvement of elite varieties by genome editing does not introduce potentially deleterious alleles from crossing and recombination.

The power of inducing site-specific DSBs

Already for classical breeding, the induction of DNA double-strand breaks (DSBs) by gamma irradiation was used to achieve genetic variability. The repair of these DSBs occurs in the large majority of cases by non-homologous end joining (NHEJ), which is error-prone [8]. It results in mutations such as deletions and insertions at the break site leading to new alleles that were not available before in the breeding population. Although most of these alleles were adverse for growth and/or yield, once and again mutations were isolated resulting in phenotypes that were attractive for breeders, such as cereals with shorter stems [9]. In the last two decades, classical transgenic approaches became available such as Agrobacterium-mediated transformation [10] or biolistic transformation [11, 12]. Thus, traits from utterly unrelated species became accessible. However, conventional mutation breeding and classical transgenic approaches are always non-specific as mutation and transgene insertion occur at random sites. Additionally, more modifications than the desired one are introduced. After it became clear that site-specific endonucleases can be used to induce DSBs in plant cells [13] resulting in directed mutagenesis of the plant genomes [14, 15], efforts were undertaken to target double strand breaks to specific genes of interest. This could be achieved by designing synthetic nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [16]. However, the generation of genetic diversity on a large scale was only enabled through the characterization of the CRISPR/Cas-system. It makes use of the Cas9 nuclease that is guided by a programmable RNA to genomic sites of interest. Compared to the time-consuming and expensive cloning procedure of ZFNs and TALENs, the RNA based sequence specificity of the CRISPR/Cas-system enables cheap and fast adaptation to various sites and provides mutagenesis at high frequencies, also for plant genomes [17,18,19,20,21]. Potential disadvantages such as lower specificity can be compensated by customized systems such as paired nickases [22,23,24] or designed Cas9 variants [25, 26], highlighting the versatility of the system. As a consequence, numerous publications elucidated its potential for targeted mutagenesis and in particular for the improvement of qualitative traits in plants (for details see current reviews: [27,28,29,30]) For a comprehensive overview on crop traits modified by genome editing, see Zhang et al. [31]. Yet, the most outstanding feature represents its multiplexing applicability. Whereas ZFNs and TALENs are barely usable for multiplexing applications, the CRISPR/Cas9-system can be easily programmed to target several sites simultaneously [32,33,34,35]. This not only allows the manipulation of numerous traits in a single generation, but also provides access to the fine-tuning and optimization of relevant traits through targeted generation of genetic diversity.

CRISPR enables immediate generation of genomic diversity for breeding

Several recent studies have demonstrated the potential of CRISPR/Cas to generate a broad range of allelic diversity at specific loci.

Shen et al. succeeded in editing eight yield or quality relevant genes in rice simultaneously [36]. Despite the high level of multiplexing, mutation rates in transgenic rice ranged from 50 to 100%. These high efficiencies allowed the isolation of mutants carrying homozygous mutated alleles of all eight targeted genes simultaneously. In addition to homozygous octuple mutants, septuple and sixtuple mutants as well as heterozygous mutants for all targeted genes were obtained. Thus, a wide range of different genotypes providing ample genetic diversity for selection could be generated within only one generation.

Another recent study showed that editing the same QTLs (Quantitative Trait Loci) can have different outcomes depending on the genetic background [37]. Two QTLs regulating grain size (GRAIN SIZE3, GS3) and grain number (Grain number 1a, Gn1a) were edited in five different widely cultivated rice varieties. Loss-of-function mutations in these QTLs were described to enhance yield [38, 39]. The authors report very high mutagenesis efficiency, which prevented the isolation of Gn1a single mutants, only allowing GS3/Gn1a double mutants and GS3 single mutants to be isolated. Surprisingly, seven of the ten novel genotypes had decreased grain yield compared to the WT, indicating strong dependency of the editing outcome on genetic background and highlighting the utility of genetic diversity across different backgrounds.

Zhou et al. achieved simultaneous editing of three yield related QTLs in elite rice backgrounds [40]. They targeted the same two QTLs, GS3 and Gn1a, in addition to GRAIN WIDTH and WEIGHT 2 (GW2). All combinations of biallelic or homozygous single, double and triple mutants were obtained. The triple mutants showed increases in the yield related traits panicle length, flower number per panicle as well as grain length, width and weight. Unlike the study from Shen et al. [37], the resulting yield related phenotypic effects of the triple mutants were consistent across all 3 varieties employed in the study. This suggests that simultaneous disruption of these three genes could be used as a simple, generally applicable “formula” for yield increase in different varieties. However, for one of the three varieties the triple mutant showed a semi-dwarf phenotype, again suggesting background specific pleiotropic effects.

The multiplexing capability of CRISPR combined with its high efficiency in rice could recently be harnessed to create a system enabling the clonal reproduction from F1 hybrids, thus preserving the favourable high degree of heterozygosity [41]. Simultaneous targeting of three meiotic genes resulted in replacement of meiosis by a mitosis-like cell division generating clonal diploid gametes and tetraploid seeds. To prevent increase in ploidy, additional targeting of a gene involved in fertilization (MATRILINEAL), induced generation of clonal diploid seeds from hybrids that stably preserved heterozygosity.

As highlighted by another recent study, the polyploid nature of many crops can be a valuable source of genetic diversity [42]. The oil profile of the hexaploid oilseed crop Camelina sativa is dominated by polyunsaturated fatty acids and the development of new varieties rich in monounsaturated fatty acids is desirable. By targeting all three homeologs of the CsFAD2 (Fatty Acid Desaturase 2) gene involved in fatty acid metabolism, a diverse set of genetic combinations with single, double and triple knockouts could be generated. The obtained lines varied strongly in their lipid profiles, with monounsaturated fatty acid levels in the oil ranging from 10%, like in wild type, up to 62% in homozygous triple mutants. As complete mutants with the strongest change in oil profile showed growth defects, the large mutant diversity could then be used for genetic fine-tuning of the trait, combining improved oil profile without growth defect.

Creating new diversity in regulatory elements to generate a range of dosage effect alleles

Cis-regulatory elements are noncoding DNA sequences that contain binding sites for transcription factors or other molecules influencing transcription, the most common examples being promoters and enhancers. Promoters are generally bound by a common set of conserved transcription factors. In contrast, enhancers are much more variable. They can be located remote from the regulated gene and not only upstream but also downstream and even in introns [43]. Furthermore, enhancers are able to physically interact with target genes by altering chromatin state [44]. This regulatory part of the genome received much less attention than protein coding sequences in the past. However, several recent publications have demonstrated the enormous potential for crop improvement by editing regulatory sequences (see also [45]). Whereas classical knock-out mutations usually mediate complete loss-of-function with accompanying pleiotropic effects [46], editing regulatory elements offers the possibility to generate a range of alleles with varying expression intensity for precise fine-tuning of gene dosage (see Fig. 1).

Fig. 1 Editing of cis-regulatory elements for the generation of dosage effect alleles. In contrast to conventional editing of coding sequences, editing of cis-regulatory elements enables the fine-tuning towards optimal gene expression level. Red colour indicates repressive, green colour activating transcription factors. Red Triangles indicate CRISPR cleavage sites. Orange sections indicate CRISPR/Cas-induced mutations Full size image

In this regard, the Lippman lab at CSHL has recently achieved pioneering breakthroughs. First, they achieved optimization of inflorescence architecture in tomato by generating new weak transcriptional alleles [47]. They improved inflorescence architecture by combining two natural mutations mediating reduced expression of the tomato homologs of the Arabidopsis genes SEPALLATA4 and FRUITFULL. The improved inflorescence architecture increased fruit number and weight as well as yield without a concomitant reduction in sugar content. Importantly, optimal inflorescence architecture could only be realized by a moderate increase in branching, which was dependent on alleles supporting reduced expression, one of them being in a heterozygous state. In contrast, combining CRISPR/Cas-mediated complete KO alleles in a homozygous state resulted in excessively branched inflorescences that produced infertile flowers. However, by targeting Cis-regulatory elements of above-mentioned genes with CRISPR, they generated a range of new alleles supporting varying expression levels for optimization of inflorescence architecture. The authors also identified a further promising Cis-regulatory element as editing target, LIN, which is another tomato SEPALLATA4 homolog. Alleles conveying reduced LIN expression might enable subtle increases in flower production. The fact that rice carries a homolog of LIN that controls panicle architecture and grain production [48] suggests that the approach might be extended to other crop species.

Following this, the same group further developed this approach to a generally applicable genetic scheme for rapid generation and evaluation of novel transcriptional alleles [49]. In this system, a biallelic mutant is generated of the gene for which novel transcriptional alleles are desired. This mutant is transformed with a multiplex CRISPR system targeting the promoter of the gene of interest at many sites and crossed with WT. The progeny from the cross inherits one WT and one mutated allele that can be edited by Cas9. As the second allele is mutated, the transcriptional effect of novel mutations in the WT allele are immediately exposed in the phenotype. In the next generation, the transgene can be segregated out and novel transcriptional alleles can be fixed immediately, generating a population showing a wide variation of expression levels for the gene of interest in a transgene-free background. The broad feasibility and usefulness of this approach was demonstrated by applying the system to three genes regulating fruit size, inflorescence branching, and plant architecture. In all cases, a strong level of dosage sensitivity was observed. More strikingly, the relationship between gene dosage and phenotypic outcome was sometimes non-linear, indicating complex interactions in case of dose-sensitive developmental genes that function in complex regulatory networks [50], which further highlights the potential of targeting the promoters of other developmental regulators to modify diverse traits [49].

Fine-tuning of gene expression can also be achieved by targeting upstream ORFs (uORFs), short protein-coding elements located in the 5’UTR of an mRNA, upstream of the main ORF. Usually, uORFs act as post-transcriptional inhibitors of translation of the downstream pORF. They are quite widespread, in plants, around 30–40% of genes exhibit uORFs [51]. Now, the Gao lab demonstrated that CRISPR mediated disruption of uORFs can be used as a generally applicable means for increasing the production of a specific protein by enhancing translation of the respective mRNA [52]. In reporter gene assays, protein activity could be enhanced 8-fold by uORF disruption. The strategy also proved successful when it was applied to 4 different endogenous uORFs, two in Arabidopsis and two in lettuce. Agronomic relevance could also be shown by disruption of the uORF of LsGGP2, which encodes a key enzyme in vitamin C biosynthesis in lettuce. uORF disruption increased foliar ascorbic acid content by 157% and enhanced tolerance against oxidative stress.

Opening up the genetic diversity from uncultured species

There are over 300,000 plant species. Less than 200 are used commercially, and only 3 species, wheat, rice and maize, provide most of the energy for human consumption [53, 54]. Further modification and improvement of elite varieties may not always be the most prudent path for generating new varieties adapted to altering conditions. In order to generate crops with novel properties, it could be highly useful to open up the enormous genetic diversity present in wild species or uncultured varieties from elite crop species by rapid domestication using genome editing. This applies especially to improvement of complex polygenic traits such as abiotic stress tolerance [55]. During the process of crop domestication, different crops have been selected for analogous traits such as favourable plant architecture and simultaneous flowering for simple harvest or large fruits for high yield. Our understanding of the genetic basis for these domestication traits is growing steadily and an increasing number of so-called domestication genes have been identified [54]. By targeting these genes with CRISPR, the domestication process can be accelerated dramatically. This is now finally possible, as demonstrated by three recent studies.

Zsögön et al. report de-novo domestication of the ancestral tomato relative Solanum pimpinellifolium, which exhibits a high degree of stress-tolerance [56]. Much of the genetic basis for stress tolerance was lost during the long domestication process of tomato. They used a multiplex CRISPR/Cas9 approach for simultaneous functional disruption of six domestication genes involved in plant architecture, yield components and nutritional quality. As in the other studies involving multiplex gene editing in tomato, efficiencies were extremely high since only mutated alleles were recovered. Compared to the wild parent, fruit size could be increased threefold and fruit number tenfold in a single generation and within a single transformation experiment. Furthermore, fruit shape was improved and nutritional quality enhanced by increasing lycopene content twofold, which translates to a fivefold increase compared to our modern cultivated tomato.

In the same issue of Nature Biotechnology, Li et al. report a similar approach for de-novo domestication of four wild tomato accessions each offering genetic diversity for resistance against specific stress conditions like bacterial spot disease or salt stress [57]. Using the multiplex capability of CRISPR, they simultaneously edited 4 target sites involved in plant architecture (SP; SELF PRUNING), flowering time (SP5G; SELF PRUNING 5G) and fruit size (SlCLV3; CLAVATA3 and SlWUS; WUSCHEL), in all four accessions (see Fig. 2). In addition to targeting coding regions for loss-of-function mutations, they also targeted regulatory regions to generate weak transcriptional alleles. In the case of SP and SP5G, more than 100 mutated alleles were created allowing a continuum of flower production, fruit production and architecture to be generated within one generation. In contrast to Zsögön et al., who could only recover completely mutated plants due to high efficiency, Li et al. observed the whole range of combinations from only one mutated gene to all four genes mutated. The completely edited plants exhibited earlier and synchronized flowering, determinate growth architecture and increased fruit size, while retaining their original stress resistance.

Fig. 2 De-novo domestication of tomato by CRISPR/Cas9-mediated multiplex editing. By simultaneously editing four genes involved in plant architecture (SP), flowering time (SP5G) and fruit size (SlCLV3 and SlWUS), Li et al. [57] achieved accelerated domestication of wild tomato. Figure design according to Li et al. [57] Full size image

More recently, rapid improvement of domestication traits hinting at de-novo domestication were undertaken in an orphan crop of the Solanaceae family, Physalis pruinosa, a striking achievement considering the previous lack of reference genome, gene annotation data and transformation protocol [58]. Initially, genomic resources had to be generated by whole genome sequencing and RNA sequencing de-novo assemblies, which subsequently enabled the identification of orthologues of domestication genes known from other Solanaceae crops. Three such genes were chosen as targets for genome editing, the Physalis pruinosa orthologues of SP, SP5G and CLAVATA1 (SlCLV1). SP is a flowering repressor and weak alleles provide a compact determinate growth that enables simple mechanized harvesting. However, the effect from CRISPR generated null alleles of Ppr-sp was too strong, limiting fruit production similar to the null sp allele in tomato, where a weak transcriptional allele is optimal. SP5G was identified recently as an important domestication gene since null alleles eliminate day length sensitivity in tomato and other crops [59]. Concerning flowering, CRISPR Ppr-sp5g mutants did not show a useful effect. However, the mutants showed moderate shoot termination resulting in higher fruit amount along each shoot. The Physalis orthologue of CLV1 was chosen as target for its involvement in the CLAVATA-WUSCHEL meristem size pathway influencing fruit size. Weak transcriptional CLV3 alleles mediate enlarged fruits in many crops, whereas clv3 null alleles mediate excessive and disorganized fruit production. Since CLV1 acts as one of several redundant CLV3 receptors, clv1 null alleles might mimic weak transcriptional CLV3 alleles. Indeed, the resulting Ppr-clv1 mutants showed a 24% increase in fruit mass.