Glossary on genome editing

A method whereby an Agrobacterium tumefaciens solution containing plasmid is infiltrated in plant tissue (mainly leaves) so as to induce a high and transient expression of tDNA.

Gene of a sexually compatible plant

Random integration of a cisgene in an abnormal location of the plant genome

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

An adaptive immune system of bacteria and archaea, used as a method of genome editing. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) describes a segment of the host genome that contains repetitive sequences and a special sequence segment of viral DNA (called spacer). Proteins associated with CRISPR use these specific sequences for molecular localisation, to protect the cell by finding and destroying viral DNA.

A protein (endonuclease) in the CRISPR-based bacterial immune system, used for genome editing. Cpf1 uses an RNA molecule to localise 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.

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

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 were also fused with nuclease-deactivated Cas9 (dCas9, d = deficient). The presence of a dimer is therefore also required to make a double-strand break.

The term includes several new methods, which are characterised by specific similarities. These are that targeted and localised double-strand breaks are induced in the DNA. Due to the imprecise DNA repair mechanism (NHEJ: non-homologous end-joining), double-strand breaks may result in mutations (InDels, SNPs). In the presence of a repair model (known as the template), the 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.

Short RNA segments that guide the DNA cleavage enzyme (endonuclease) to the target location in the genome. gRNAs contain the match site to the target sequence (usually 20 bases) and a segment that interacts with the endonuclease (e.g. Cas9).

Repair mechanism of the cell for DNA strand breaks, which consists of a segment of homologous donor DNA (acting as a repair template) “patching” the DNA. The 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.

Abbreviation for insertion and/or deletion. Describes the random removal or introduction of nucleotides in the DNA sequence. InDels occur when double-strand breaks are made in the DNA, which are inaccurately repaired by NHEJ. This often leads to the displacement of the open reading frame and/or the creation of premature stop codons and therefore a break in the gene.

Any new combination of genes and regulatory elements of the same kind or sexually compatible plants, which are recombined outside of the organism and introduced into the plant genome, where they can be passed on.

Process that leads to mutations in the DNA sequence.

Change in the DNA sequence of an organism. The change can be small (SNPs, small InDels), include thousands of base pairs (insertions, deletions, translocations, substitution), or affect entire chromosomes.

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).

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 random inclusion or removal of nucleotides around the break site, which results in insertions or deletions in the genetic code.

Direct or indirect, inadvertent, short- or long-term consequence of an effect on an organism, which differs from the intended aim - for example, when a genome editing nuclease cleaves DNA at a random, "off-target" site, which is similar to the intended target site.

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.

This term includes innovations such as genome sequencing, ‘omics and methods to expand the 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.

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.

Created by joining two or more nucleic acid molecules together outside of the organism, which are then passed on after inclusion in the organism (see BVL, 2017; EFSA, 2015; NIH, 2016).

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.

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.

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

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).

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).

Exchange of a single nucleotide in the DNA sequence.

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 ZNFs. SDN is subdivided into SDN-1, SDN-2 and SDN-3, depending on the degree of induced change.

Method of genome editing whereby a domain of the protein recognises 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.

Stable integration of DNA of not sexually compatible species in a plant genome.

Method of genome editing whereby a domain of the protein recognises a specific DNA sequence and another domain cleaves this DNA. ZNFs 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.