Glossary - Genome editing - Breeding methods - Innovation

Agroinfiltration

A method wherein a plasmid carrying Agrobacterium tumefaciens solution is infiltrated in plant tissues (often leaves) to trigger a high and transient expression of the transfer DNA.

Allele

A variant form of a gene at a particular locus on a chromosome. Different alleles produce variations in inherited characteristics.

Base editing is a genome editing method based on CRISPR/Cas9. It allows the targeted and directed conversion of one DNA or RNA base into another (i.e. the introduction of a point mutation) using DNA or RNA base editors. These base editors comprise a catalytically disabled nuclease fused to a catalytically active nucleobase deaminase enzyme.

Chromosome

A thread-like structure that contains a single length of DNA, usually carrying many hundreds of genes. Different plants have different number of chromosomes: for example, sugarbeet has 18 chromosomes. Plants also vary in terms of ploidy, i.e., the number of sets of chromosomes.

Chromosomes usually reside in the nucleus of a cell. Besides chromosomes, also the plants’ organelles (mitochondria and chloroplasts) contain DNA.

Cisgene

Gene of a sexually compatible plant

Cisgenesis

Integration of a cisgene in the plant genome.

Cisgenesis sensu stricto

The replacement or addition (due to naturally occurring presence/absence variation) of a cisgene/allele to the original location in the genome.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

An adaptive component of bacterial and archaeal immune systems, used as a method of genome editing. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) describes a segment of repetitive sequences separated by (foreign) viral DNA (called spacer). Proteins associated with CRISPR (Cas) use these specific sequences to protect the cell by finding and destroying viral DNA.

Cpf1

A protein (endonuclease) in the CRISPR-based bacterial immune system used for genome editing. Cpf1 uses an RNA molecule to localize complementary DNA (guide RNA) and cleave it, resulting in a double-strand break. Compared to Cas9, Cpf1 has a slightly different PAM-sequence and a different cleavage pattern.

CRISPR associated protein 9 (Cas9)

A protein (endonuclease) in the CRISPR-based bacterial immune system used for genome editing. Cas9 uses an RNA molecule to localize complementary DNA (guide RNA) and cleave it, resulting in a double-strand break. In CRISPR-mediated immune defense, cleaving viral DNA prevents infection of the host cell.

Donor organism

The organism from which genetic material is obtained for transfer to the recipient organism.

Edit

A change to the DNA sequence (e.g. insertion, deletion, substitution) at a specific site in the genome that is produced with targeted mutagenesis methods of genome editing and results in an intended effect (e.g. gene disruption).

Enzyme

A protein acting as a biological catalyst for chemical reactions.

FokI

Protein (endonuclease) that has been bound to the DNA-binding domains zinc finger (ZF) or transcription activator-like effector (TALE). FokI only cleaves a single strand of DNA (single-strand break); therefore, a pair of ZFNs or TALENs is needed to make double-strand breaks. The FokI cleavage domains are also fused with nuclease-deactivated Cas9 (dCas9, d = deficient). The presence of a dimer is therefore also required to make a double-strand break.

Gene

A functional unit of heredity that is a segment of DNA in a specific part of a chromosome. A gene typically directs the formation of a protein or RNA molecule.

Gene pool

Germplasm (i.e. genetic material) within which sexual recombination is possible as a result of hybridization, including via methods such as embryo culture.

Genetically Modified Organism GMO

Biological organism whose genetic material has been changed in such a way, which would not be possible under natural conditions like crossing or natural recombination.

Genome

The complete DNA of an organism. Plant genomes are highly variable in size.

Genome Editing

The term includes several new methods, with which targeted and site-specific double-strand breaks are induced in the DNA. Due to the cell`s own but often error-prone DNA repair mechanism (NHEJ: non-homologous end-joining), double-strand breaks may result in mutations (InDels, SNPs). In the presence of a repair template, the (cellular) precise mechanism of homologous recombination (HR) can be used to implement specific changes in the DNA or to insert entire genes. The methods grouped under this term are CRISPR/Cas, TALENs, ZFNs, meganucleases and ODM.

Genotype

The genetic constitution of an organism.

Guide RNA (gRNA)

Short segments of RNA used to direct the DNA cutting protein to the target location in the genome. gRNA segments contain the region of homology to the target sequence (usually 20 bases), and a sequence that interacts with the nuclease (e.g. Cas9).

Heterozygous

Having different variants (alleles) of a specific gene on the homologous chromosomes of a cell or an organism.

Homologous recombination (HR)

Repair mechanism of the cell for DNA strand breaks which uses a segment of homologous donor DNA (acting as a repair template), “patching” the DNA. The repair template donor DNA must have major similarities to the cleavage sites of the DNA in its sequences (homologies) to be able to be inserted. HR is a more precise repair than NHEJ. During genome editing, the donor DNA is designed and inserted in a targeted manner; hence, it could potentially replace a pathological gene with a healthy copy.

Homozygous

Having the same variant (allele) of a specific gene on homologous chromosomes of a cell or an organism.

Inbred line

A line that has been selfed to sufficient homozygosity to be considered homozygote.

InDels

Abbreviation for insertion and/or deletion. Describes the random removal or introduction of nucleotides in the DNA sequence. InDels occur when DNA double-strands are inaccurately repaired by NHEJ. This often leads to the disruption of the open reading frame and/or the creation of premature stop codons and therefore a loss of gene function.

Intragenesis

Any new combination of genes and regulatory elements of that same species that were combined outside the organism and incorporated into the plant genome where they are capable of continued propagation.

Meganuclease

A type of enzyme that binds to and cuts DNA at specific DNA sequences. Meganucleases can be used in genome editing for both nonhomologous end joining (NHEJ) and homology directed repair-mediated alterations.

Mutagenesis

Process that leads to mutations in the DNA sequence.

Mutation

A change of an organism´s own DNA sequence. The change can be small (SNPs, small InDels) or can span thousands of base pairs (insertions, deletions, translocations, replacements) or can affect whole chromosomes but is limited to sequence variations within the gene pool (crossable species).

New breeding methods

This is an umbrella term for new methods of plant breeding that create very precise changes and variability in plants. This precision and efficiency sets them apart from conventional breeding methods such as crossing, selection and traditional mutagenesis using chemicals or radiation. This term includes: agroinfiltration, cisgenesis, CRISPR/Cas, grafting, ODM (oligo-directed mutagenesis), RdDM (RNA-dependent DNA methylation), reverse breeding, TALENs and ZFNs (zinc finger nucleases).

Non-homologous end-joining (NHEJ)

DNA repair mechanism of the cell that works by linking the free ends after a DNA strand break. Compared to homologous recombination, this mechanism is imprecise and often leads to the random inclusion or removal of nucleotides around the break site, which results in insertions or deletions in the genetic code.

Nuclease

An enzyme that can cut through DNA or RNA strands.

Off-target effect

A direct or indirect, unintended, short- or long-term consequence of an intervention on an organism other than the intended effect on that organism. For example, when a genome editing enzyme cuts DNA at an unintended, "off-target," site that is similar to the intended target.

Oligo-directed mutagenesis (ODM)

Method of genome editing whereby an oligonucleotide, usually 20–100 nucleotides in length, is used to effect a single nucleotide exchange in the plant genome. Apart from one modified base pair, the oligonucleotide is identical (homologous) to the target sequence in the plant genome.

Phenotype

Different observable characteristics that can be produced by two or more alleles of a genetic locus within a species that control a trait which is the result of the underlying genotype and its interaction with the environment. For example, flower colour is a trait, and red and white flower colours are two different phenotypes for the flower colour trait.

Plant breeding innovations (PBI)

This term includes innovations such as genome sequencing, omics and methods to expand genetic variation. They help increase our knowledge in the field of plant genetics, physiology and plant-pathogen interaction. Methods under this umbrella term help plant breeders to discover and understand important characteristics for breeding and to achieve breeding objectives faster and more efficiently.

Plasmid

In nature, a plasmid is a small DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently from genomic DNA. Plasmids are most commonly found as small circular, double-stranded DNA molecules in bacteria. In genetic engineering, isolated, artificial plasmids are widely used as vectors in molecular cloning.

Prime editing

Prime editing is a CRISPR/Cas9-based system that rewrites DNA to induce targeted and precise insertions, deletions, and base-to-base conversions. This method uses a prime editing guide RNA (pegRNA), a catalytically impaired Cas9 nuclease, and an engineered reverse transcriptase that initiates the precise change, addition or removal of a sequences. Once the new genetic material is incorporated into the cut strand of DNA, the prime editor nicks the unedited strand, signalling to the cell to rebuild it to match the edited strand.

Protospacer adjacent motif (PAM)

Short nucleotide sequence that must be present directly next to the DNA target sequence, so Cas9 or Cpf1 can bind and cleave them. The absence of PAM in the host genome prevents the cleavage of the CRISPR area.

Recombinant nucleic acid

Produced by joining two or more nucleic acid molecules by whatever means outside an organism capable of continued propagation after incorporation into an organism (in accordance to BVL, 2017).

Repair template

A nucleic acid sequence used to direct cellular DNA repair pathways to incorporate specific DNA sequence changes at or near a target site.

Reverse breeding

Suppression of recombination by means of an included transgene (RNAi-based) to generate fully homozygotic doubled haploid plants for hybrid breeding. The resulting plants no longer carry the transgene.

Ribonucleic acid (RNA)

A single-stranded molecule that transmits and regulates the DNA’s instructions for the development, functioning, and reproduction of all living organisms.

RNA-dependent DNA methylation (RdDM)

RdDM-induced deactivation (e.g. transcriptional gene silencing) of target genes through the methylation of promoter sequences. To induce RdDM, RNAi-based constructs are used to include sequences in the cells, which encode RNA homologously to the desired promoter region.

RNA interference (RNAi)

RNAi is a naturally occurring mechanism for gene silencing, triggered by double-stranded RNA (dsRNA). RNAi processes can be used by researchers to block, or silence, the expression of a given gene.

SDN-1

Methods whereby the achieved changes are created by random mutations (deletions, insertions or SNPs) as a result of an inaccurate DNA repair mechanism (NHEJ).

SDN-2

In the case of SDN-2, a repair model (template) is inserted into the cell, which induces small and specific changes through the precise mechanism of homologous recombination (HR).

SDN-3

In the case of SDN-3, repair of double-strand breaks through homologous recombination in connection with an inserted DNA molecule (template) is used to integrate or exchange genes and/or regulatory elements (e.g. allele variants).

Single nucleotide polymorphisms (SNPs)

Exchange of a single nucleotide in the DNA sequence.

Site-directed nucleases (SDN)

Methods which induce double-strand breaks at specific and defined points of the genome by means of endonucleases. The DNA-binding domain can be either a protein or RNA. Methods that fall under this umbrella term are CRISPR/Cas, TALENs and ZFNs. SDN is subdivided into SDN-1, SDN-2 and SDN-3, depending on the degree of induced change.

Targeted mutagenesis

The process of generating a mutation at a predefined location within a genome by using genome editing methods. The resulting mutation can be random (by using SDN-1) or specific (by using e.g., Base Editing, SDN-2 or SDN-3). Sequence insertions and replacements through targeted mutagenesis are limited to sequence variations within the gene pool (crossable species). The obtained changes could have been the result of traditional breeding or of a natural mutation.

Target sequence

A nucleic acid sequence subject to intentional binding, modification, or cleavage.

Trait

An observable (able to be seen or otherwise identified) characteristic of an organism. For example, flower colour is a trait.

Transcription activator-like effector nucleases (TALENs)

Method of genome editing whereby a domain of the protein recognizes a specific DNA sequence and another domain cleaves this DNA. TALENs consist of a succession of small DNA-binding domains that are able to detect a longer DNA sequence. This DNA-binding domain is fused with a fokI nuclease, which cleaves the DNA.

Transgenesis

Stable integration of DNA of sexually incompatible species in a plant genome.

Transgenic organism

An organism into which one or more genes have been transferred or introduced from another species of organism (GMO).

Variety

A distinct, uniform, stable genotype approved for its value for cultivation and use.

Zinc finger nucleases (ZFNs)

Method of genome editing whereby a domain of the protein recognizes a specific DNA sequence and another domain cleaves this DNA. ZFNs consist of a succession of small DNA-binding domains that are able to detect a longer DNA sequence. This DNA-binding domain is fused with a nuclease, which cleaves the DNA.

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