Molecular Scissors Revolutionize Plant Breeding

Genome Editing Using the CRISPR/Cas System

  • Fig. 1: Programmed Cas9-mediated DSB induction and repair. (A)Fig. 1: Programmed Cas9-mediated DSB induction and repair. (A)
  • Fig. 1: Programmed Cas9-mediated DSB induction and repair. (A)
  •  A 20nt long part of the gRNA determines the position in the genome, at which Cas9 binds and induces a DSB. As the target sequence recognition is mediated specifically via RNA-DNA base pairing, Cas9 can be programmed for nearly every target in a genome. The induced DSB is repaired by cellular mechanisms: the conservative HR utilizes identical sequences from ectopic regions of the genome, the homologous chromosome or the sister chromatid for repair. However, the prevalent mechanism in higher eukaryotes is the joining of broken ends via NHEJ, which often results in small insertions or deletions. (B) The targeted introduction of a DSB in a plant genome can be applied for site-specific mutagenesis via the NHEJ repair pathway (SDN-1). Alternatively, the simultaneous delivery of a linear DNA sequence with homologies to the broken ends in parallel to DSB induction can be applied to introduce small (< 20nt, SDN-2) or larger sequence stretches (SDN-3) from the same or other species by HR-mediated repair (Gene Targeting) [5]. SDN: Site Directed Nuclease; classification.
  • Fig. 2: Genome Engineering. (A)
  • The application of inactivated Cas9 orthologs (dCas9) with different specificities (gRNA A1-An, gRNA B1-Bn) and fused effectors allow for targeted modulation of gene expression. (B) The utilization of Cas9 orthologs with different target sequences enables the simultaneous induction of DSBs at several loci. This way, the limitations of classical plant breeding can be overcome, as genomic sequences can be (a) inverted, (b) removed or mobilized and reciprocal chromosome translocations be induced (c1, c2) to create new desirable or to break undesirable genetic linkages. Sequences of the same species can thus be combined to new functional units and can therefore support attempts to better exploit the genetic potential of a species.
The bacterial CRISPR/Cas system revolutionizes the possibilities of targeted genome modifications and thus enables unprecedented progress in plant breeding. The understanding of natural DNA repair mechanisms in combination with this highly specific, versatile and efficient molecular tool allows for both the precise introduction of mutations in DNA and targeted reshuffling of genomes. Such modifications of genetic information can no longer be distinguished from naturally occurring events.
DNA is continuously exposed to damaging factors that can cause mutations. As the correct transmission of the genetic information to the offspring is essential, highly specific repair mechanisms evolved during the evolution of life. However, mutations not only pose a threat but can also be understood as an opportunity to better adapt to continuously changing environmental conditions and the selection pressure thereof, by allowing for improved properties. Of course, humans were by far not considering such thoughts when changing from being nomadic hunters to a sessile life-style more than 10,000 years ago. Nevertheless, those early farmers started selecting plants with superior traits or performance and thus unconsciously laid the basis for plant breeding. Later on, sexual crosses increased the breeding potential significantly, followed by the application of unspecific mutagenesis to modify crop genomes to obtain new and advantageous traits during the 20th century. Nowadays, mankind faces the challenge to find sustainable ways of meeting the nutritional demands of the continuously growing population, while land suitable for growing crop plants is finite. Thus, new approaches in plant breeding are required [1].
The CRISPR/Cas System and its Applications
So far, breeding methods to increase yield or to generate new resistances are based on the application of mutagens like Ethylmethanesulfonate (EMS) or ionizing radiation which induce unspecific mutations that subsequently need to be screened for beneficial traits. The disadvantage of this approach is that a large population needs to be treated and screened in a labor-intensive and expensive way to generate a beneficial trait, while countless unidentified mutations with unknown consequences are created in parallel.

Even though new marker assisted methods based on complete genome sequences can accelerate plant breeding, these procedures still have their limitations. The CRISPR/Cas system has the potential to overcome those hurdles and thus allows for a revolution in plant breeding.

CRISPR/Cas has been identified as an adaptive immune system of many bacteria and archaea species. In 2012, its mode of action was for the first time described by the groups of Emmanuelle Charpentier and Jennifer Doudna. They could demonstrate that bacteria can recognize intruding DNA of bacteriophages and cut them into pieces of 20 nucleotides (nt) in length, followed by their integration into a so called CRISPR (clustered regularly interspaced short palindromic repeat) locus within their genome. The latter can be understood as a kind of data cache for foreign DNA. Whenever a bacterium is confronted again with a bacteriophage with the same specific sequence, the Cas9 nuclease utilizes the 20nt recognition sequence (gRNA) to identify the intruding DNA as foreign, followed by subsequent cleavage. Consequently, the bacteriophage cannot infect the bacterium anymore. It didn’t take long for Charpentier and Doudna to understand the potential of their discovery as an unprecedented tool for molecular biology. In contrast to molecular scissors used so far to introduce site-specific breaks in genetic information, no time-consuming efforts are required anymore: Cas9 can be programmed for almost every target in a given genome by supplying a gRNA of choice. The intrinsic nuclease activity of Cas9 can thus be exploited to specifically induce targeted DNA double-strand breaks (DSBs, fig. 1A) [2]. 
DSBs are processed and removed by cellular DNA repair mechanisms. The knowledge of these processes and the factors involved allows scientists to influence the preference of the DSB repair pathway, either Non-homologous End-joining (NHEJ) for site-specific mutagenesis or Homologous Recombination (HR) to facilitate the targeted insertion of minimal sequence modifications or larger genetic sequences of the same or foreign species into the genome via Gene Targeting (fig. 1B) [3]. For example, NHEJ-mediated Cas9-induced site-specific mutagenesis of three homoeoalleles in the hexaploid wheat genome has been applied to obtain a resistance to powdery mildew [4]. Furthermore, a modification of Cas9 allows for the removal of the nuclease activity (dCas9) and fusing effectors to it via short amino acid linkers in order to modulate gene expression at desired genomic loci (fig. 2A). Due to its natural capability to recognize several target sequences simultaneously, the CRISPR/Cas system can be exploited also for biotechnological multiplexing applications. Experimentally, this can be achieved by providing several different gRNAs that are all recognized and utilized by the same Cas9. Thus, several DSBs can be introduced into a genome simultaneously to generate new desirable linkage groups or to break those between undesirable linked loci (fig. 2B). Meanwhile, several new CRISPR/Cas systems have been described in various bacterial species. Those orthologs only utilize their species-specific gRNA [6]. Therefore, positive and negative transcription-modulating effectors can be applied in parallel to DSB induction at several positions within one genome at the same time. Thus, in addition to customized genome modifications, a transient or permanent modulation of the transcriptome is also feasible [7, 8]. 
The biotechnological application of Cas9 and its orthologs allows for the targeted introduction of one or more DSBs in the plant genome. Using the natural cellular DSB repair pathways, precise mutations or genome modifications can be obtained. The versatility of the system additionally enables the targeted modulation of gene expression. This way, the natural gene pool of plant species can be exploited much better and crop traits can be optimized by customized gene expression.
Michael Pacher1 and Holger Puchta1
1 Karlsruhe Institute of Technology (KIT), Botanical Institute, Molecular Biology and Biochemistry, Karlsruhe, Germany
Director of the Botanical Institute
Karlsruhe Institute of Technology (KIT)
Botanical Institute
Molecular Biology and Biochemistry
Karlsruhe, Germany

[1] Pacher M & Puchta H (2017) From classical mutagenesis to nuclease‐based breeding – directing natural DNA repair for a natural end‐product. Plant J DOI: 10.1111/tpj.13469
[2] Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816–821
[3] Puchta H, Dujon B & Hohn B (1996) Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination. Proc Natl Acad Sci USA 93: 5055–5060
[4] Wang Y, Cheng X, Shan Q et al (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32: 947–951
[5] Fauser F, Roth N, Pacher M et al (2012) In planta gene targeting. Proc Natl Acad Sci USA 109: 7535–7540
[6] Schiml S & Puchta H (2016) Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas. Plant Methods 12: 8
[7] Steinert J, Schiml S, Fauser F & Puchta H (2015) Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus. Plant J 84:1295–1305
[8] Puchta H (2017): Applying CRISPR/Cas for genome engineering in plants: the best is yet to come. Curr Opin Plant Biol 36:1–8



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