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Summary

Although genome editing technology is being actively utilized, there is no standardization in terms and definitions for this field, which can lead to misinterpretation and miscommunication of concepts, data, and results.

Standards in the field of genome editing will harmonize and accelerate effective communication, technology development, qualification, and evaluation of genome editing products. This document is expected to improve confidence in and clarify scientific communication, data reporting, and data interpretation in the genome editing field.

This lexicon was developed to provide a unified reference set of terms and technical definitions that standardizes their use and meaning to serve the needs of the biotechnology community.

It is recognized that in rare instances exceptions may exist for some definitions within specific applications.

Description

The definitions are worded with the intention that additional context may be added with supplementary language when they are used. It is also recognized that genome editing is a rapidly evolving biotechnology and additional terms and definitions will be needed as genome editing technologies mature.

ORGANIZATION OF THE LEXICON

1. Genome editing concepts

2. Genome editing tools

    2.1 General

    2.2 CRISPR specific

    2.3 Meganuclease specific

    2.4 megaTAL specific

    2.5 TALEN specific

    2.6 ZFN specific

3. Genome editing outcomes

Terms within categories are listed alphabetically. In the Genome editing tools section, the sub-category “General” contains terms that apply to all types of genome editing tools. Additional sub-categories contain terms specific to the sub-category of genome editing technology: “CRISPR specific”, “Meganuclease specific”, “TALEN specific”, “megaTAL specific” and “ZFN specific”. A glossary listing all terms alphabetically precedes the Terms and definitions.

Glossary of terms in alphabetical order

Term

Term number

Cas nuclease

2.2.1

Cas nuclease target site

2.2.2

CRISPR associated nuclease

2.2.1

CRISPR RNA

2.2.3     

CRISPR target strand

2.2.8

crRNA

2.2.3

Cys2His2 zinc finger

2.6.2

DNA, RNA, or epigenome edit

3.1

DNA, RNA, or epigenome intended edit

3.4

DNA, RNA, or epigenome unintended edit

3.7

edit

3.1

gene editing

1.1

genome editing

1.2

genome editing off-target

1.4

genome editing target

1.5

genome editing target specificity

1.6

genome engineering

1.3

gRNA

2.2.4

guide RNA

2.2.4

HDR

3.2

homology-directed repair

3.2

InDel mutation

3.3

intended edit

3.4

meganuclease

2.3.1

meganuclease linker

2.3.2

meganuclease single chain

2.3.3

meganuclease target site

2.3.4

megaTAL

2.4.1

megaTAL linker

2.4.2

megaTAL target site

2.4.3

microhomology-mediated end joining repair

3.5

MMEJ

3.5

NHEJ

3.6

non-homologous end joining

3.6

off-target

1.4

PAM

2.2.5

protospacer adjacent motif

2.2.5

repair template

2.1.1

repeat variable diresidue

2.5.1

ribonucleoprotein

2.2.6

RNP

2.2.6

RVDs

2.5.1

sgRNA

2.2.7

single-guide RNA

2.2.7

site-directed DNA modification enzyme

2.1.2

site-directed nuclease

2.1.3

specificity

1.5

TALEN

2.5.2

TALEN linker

2.5.3

TALEN target site

2.5.4

target

1.6

target strand

2.2.8

tracrRNA

2.2.9

trans-activating CRISPR RNA

2.2.9

unintended edit

3.7

ZFN

2.6.3

ZFN linker

2.6.4

ZFN recognition helix

2.6.1

ZFN target site

2.6.6

ZFP

2.6.5

zinc finger

2.6.2

zinc finger nuclease

2.6.3

zinc finger protein

2.6.5

Terms and definitions 

1. Genome editing concepts

1.1

gene editing

techniques for genome engineering (1.3) that involve DNA repair mechanisms and/or replication for incorporating site-specific modification into a gene or genes

Note 1 to entry: Gene editing is a subclass of genome editing (1.2).

Note 2 to entry: There are a various Genome editing tools (2)

1.2

genome editing

techniques for genome engineering (1.3) that involve DNA repair mechanisms and/or replication for incorporating site-specific modification into a genomic DNA

Note 1 to entry: Gene editing (1.1) is a subclass of genome editing.

Note 2 to entry: There are a various Genome editing tools (2)

1.3

genome engineering

process of introducing intentional changes into genomic DNA

Note 1 to entry: Gene editing (1.1) and genome editing (1.2) are techniques used in genome engineering.

1.4

off-target

genome editing off-target

genomic position and/or RNA sequence distinct from the target (1.6)

EXAMPLE: off-target binding, off-target cleavage, off-target edit, off-target sequence change.

Note 1 to entry: An off-target edit is an example of an unintended edit (3.7).

1.5

specificity

genome editing target specificity

extent to which an editing agent or procedure acts only on its intended target (1.6)

Note 1 to entry: When using this term, the procedure is defined, intended target is defined, action or outcome is measured and reported, and limits of detection are reported.

1.6

target

genome editing target

nucleic acid sequence subject to intentional binding, modification, and/or cleavage

Note 1 to entry: See also off-target (1.4), Cas nuclease target site (2.2.2), meganuclease target site (2.3.4), TALEN target site (2.5.4), megaTAL target site (2.4.3), ZFN target site (2.6.6).

2. Genome editing tools

2.1 General

2.1.1

repair template

nucleic acid sequence used to direct cellular DNA repair pathways to incorporate specific DNA sequence changes at or near a target (1.6) site

2.1.2

site-directed DNA modification enzyme

enzyme capable of modifying deoxyribonucleic acids at a specific sequence

EXAMPLE: site-directed nuclease (2.1.3), site-directed adenosine deaminase

2.1.3

site-directed nuclease

sequence-specific nuclease

enzyme capable of cleaving the phosphodiester bond between adjacent bases in a nucleic acid polymer at a specific sequence

2.2 CRISPR specific

2.2.1

Cas nuclease

CRISPR associated nuclease

enzyme that is a component of CRISPR systems that that is capable of breaking the phosphodiester bonds between nucleotides

EXAMPLE: Cas3, Cas9, Cas12a, Cas13, CasX

Note 1 to entry: Some but not all Cas nucleases interact with a gRNA (2.2.4).  See also crRNA (2.2.3), sgRNA (2.2.7), tracrRNA (2.2.9)

2.2.2

Cas nuclease target site

nucleotide sequence comprising the PAM (2.2.5) and a region that hybridizes to the target sequence specific guide of a Cas RNP (2.2.6)

2.2.3

CRISPR RNA

crRNA

polyribonucleotide that includes sequence complementarity to the target (1.6) sequence and a sequence that interacts with a Cas protein and optionally tracrRNA (2.2.9)

Note 1 to entry: CRISPR RNA is a component of guide RNA (2.2.4) or a complete guide RNA, depending on the CRISPR system.

Note 2 to entry: In some CRISPR systems, a portion of the crRNA will base-pair with the tracrRNA (2.2.9) (e.g. Cas9). Other CRISPR systems lack tracrRNA (e.g. Cas12a/Cpf1). In systems that do not require tracrRNA, the guide RNA (2.2.4) is called a CRISPR RNA, or simply crRNA.

2.2.4

guide RNA

gRNA

polyribonucleotide containing regions sufficient for productive interaction with a Cas nuclease or variant to direct interaction with the specific target (1.6) sequence

Note 1 to entry: See crRNA (2.2.3), tracrRNA (2.2.9), and sgRNA (2.2.7).

Note 2 to entry: For Cas9-type proteins, the natural gRNA is comprised of a crRNA (2.2.3) that imparts sequence specificity and the tracrRNA (2.2.9) that interacts with and activates the protein. This is sometimes referred to as a dual guide. Other Cas proteins can have different gRNA structures

Note 3 to entry: sgRNAs (2.2.7) for Cas9 proteins are non-naturally occurring polyribonucleotides where the crRNA (2.2.3) and tracrRNA (2.2.9) are fused with an artificial linker.

Note 4 to entry: In some cases, chemical modifications of the polyribonucleotide are used, such as modifications to the phosphodiester linkages, bases, or sugar moieties. These can include substitution of DNA (2’-deoxy) or 2’-methoxy nucleotides for RNA nucleotides etc.

2.2.5

protospacer adjacent motif

PAM

short nucleotide motif in the targeted region of nucleic acid required for guided Cas nuclease or variant binding

Note 1 to entry: PAMs are distinct from, but in close proximity to, nucleic acid sequence targeted by guide RNA (2.2.4)

2.2.6

ribonucleoprotein

RNP

complex comprising protein bound to RNA

Note 1 to entry: In the context of CRISPR-based genome editing (1.2), RNP refers to the complex of Cas protein(s) and guide RNA (2.2.4).

2.2.7

single-guide RNA

sgRNA

fusion of crRNA (2.2.3) and tracrRNA (2.2.9)

Note 1 to entry: See guide RNA (2.2.4).

2.2.8

target strand

CRISPR target strand

single-stranded nucleic acid sequence that is complementary to the guide RNA (2.2.4) of a Cas protein or variant

2.2.9

trans-activating CRISPR RNA

tracrRNA

polyribonucleotide that base-pairs with the crRNA (2.2.3) and interacts with a Cas nuclease to enable sequence-specific interaction of the target (1.6) nucleic acid

Note 1 to entry: Trans-activating CRISPR RNA is an optional component of guide RNA (2.2.4).

2.3 Meganuclease specific

2.3.1

meganuclease

variant of the LAGLIDADG subtype of homing endonucleases engineered to recognize a 15-40 bp DNA target (1.6) sequence different than the site recognized by the parent endonuclease

Note 1 to entry: The LAGLIDADG consensus sequence represents an alpha helix that serves as a dimerization interface and key component in the DNA cleavage site in this family of meganucleases.

2.3.2

meganuclease linker

natural or artificially derived polypeptide sequence that links two LAGLIDADG domains to one another to form a single polypeptide chain

Note 1 to entry: The LAGLIDADG consensus sequence represents an alpha helix that serves as a dimerization interface and key component in the DNA cleavage site in this family of meganucleases.

2.3.3

meganuclease single chain

meganuclease (2.3.1) composed of two LAGLIDADG domains joined by either a natural or artificially derived polypeptide linker in order to be expressed as a single polypeptide chain

Note 1 to entry: The LAGLIDADG consensus sequence represents an alpha helix that serves as a dimerization interface and key component in the DNA cleavage site in this family of meganucleases.

2.3.4

meganuclease target site

DNA sequence recognized by meganucleases (2.3.1).

Note 1 to entry: Meganuclease target sites are 15-40 base pair DNA sequence consisting of two equal length half sites separated by a 4 base pair middle sequence (also known as central 4). Cleavage occurs at the junction of the half sites and the middle site on each DNA strand leaving a 4 base pair 3’ overhang.

2.4 megaTAL specific

2.4.1

megaTAL

artificial nucleases composed of an array of Transcription Activator-like (TAL) Effector (TALE) DNA binding domains, a megaTAL linker (2.4.2), and a meganuclease (2.3.1)

Note 1 to entry: For Transcription Activator-like Effector, the definition of the U.S. National Library of Medicine has been used [1].

2.4.2

megaTAL linker

amino acid sequence that links an array of TAL DNA binding domains and a meganuclease (2.3.1)

2.4.3

megaTAL target site

intended DNA binding site of a megaTAL (2.4.1), encompassing the DNA sequence targeted by both the TAL array and the meganuclease (2.3.1)

2.5 TALEN specific

2.5.1

repeat variable diresidue

RVDs

two amino acid sequence in TAL repeats that imparts DNA binding specificity

2.5.2

transcription activator-like effector nuclease

TALEN

artificial nucleases composed of an endodeoxyribonuclease fused to DNA-binding domains of TALEs that cleave DNA at a defined distance from TALE recognition sequences.

Note 1 to entry: A TALEN can refer to a pair of TALE-FokI fusion proteins that dimerize on opposite strands of DNA adjacent to a target (1.6) site for cleavage.

Note 2 to entry: For Transcription Activator-like Effector, the definition of the U.S. National Library of Medicine has been used [1].

2.5.3

TALEN linker

polypeptide sequence that links an array of TAL DNA binding domains and an endodeoxyribonuclease, typically FokI

2.5.4

TALEN target site

DNA sequence recognized by TALENs (2.5.2)

Note 1 to entry: Typical TALEN target sites are recognized by a pair of TALENs (2.5.2) and contain a central spacer region flanked by upstream and downstream sequences that are each recognized by one TALEN. This pair is designed in such a way that two TALEN nuclease domains dimerize to cleave DNA within the spacer region.

2.6 ZFN specific

2.6.1

ZFN recognition helix

seven residue positions within a zinc finger that are most directly responsible for its DNA binding preference.

Note 1 to entry: The seven residues comprise the first six residues of the alpha helix, along with the residue immediately preceding the amino terminus of the helix. They are typically referred to as positions +1 to +6 (within the alpha helix) and position (-1) (immediately preceding the helix).

2.6.2

zinc finger

Cys2His2 zinc finger

DNA binding domain (typically containing 28 amino acids) that folds via coordination of zinc into a compact structure consisting of two beta strands and one alpha-helix (β β α)

2.6.3

zinc finger nuclease

ZFN

chimeric protein consisting of an array of Cys2His2 zinc fingers (2.6.2) linked to a DNA cleavage domain

Note 1 to entry: FokI is prevalently used as the DNA cleavage domain bound to a zinc finger (2.6.2).

Note 2 to entry: Binding of two ZFNs to a pair of appropriately spaced DNA target sites enables nuclease domain dimerization and DNA cleavage between the targets.

2.6.4

ZFN linker

polypeptide sequence that links an array of zinc finger (2.6.2) binding domains and a DNA cleavage domain

Note 1 to entry: FokI is prevalently uses as the DNA cleavage domain bound to a zinc finger (2.6.2).

2.6.5

zinc finger protein

ZFP

DNA binding protein consisting of a tandem array of Cys2His2 zinc fingers (2.6.2)

2.6.6

ZFN target site

DNA sequence recognized by a pair of ZFNs (2.6.3)

Note 1 to entry: Typical ZFN target sites contain a central spacer region flanked by DNA sequences that each are recognized by an array of zinc fingers (2.6.2) oriented such that the ZFN nuclease domains dimerize and cleave within the spacer.

3. Genome editing outcomes

3.1

edit

DNA, RNA, or epigenome edit

change to genomic DNA sequence, RNA sequence, or epigenetic signature resulting from the application of genome editing (1.2) components (e.g. nuclease, repair template (2.1.1))

EXAMPLE: insertion, deletion, substitution, deamination, methylation, demethylation

3.2

homology-directed repair

HDR

mechanism of recombinational DNA repair where repair is templated by a polynucleotide with regions corresponding to sequences flanking the target (1.6) site

EXAMPLE: single-stranded DNA oligonucleotide templated HDR

Note 1 to entry: Repair templates (2.1.1) can be exogenously introduced to achieve sequence changes in genome editing (1.2) approaches.

Note 2 to entry: For recombinational DNA repair, the definition of the U.S. National Library of Medicine has been used [2].

3.3

InDel mutation

sequence change caused by the insertion or deletion of nucleotide bases

3.4

intended edit

DNA, RNA, or epigenome intended edit

designed change to the genomic DNA sequence, RNA sequence, or epigenetic signature at the target (1.6) resulting from the application of genome editing (1.2) components (e.g. nuclease, repair template (2.1.1))

Note 1 to entry: See edit (3.1)

3.5

microhomology-mediated end joining repair

MMEJ

mechanism of DNA end-joining repair where the DNA ends are rejoined to each other using short regions (typically 2-25 base pairs) of homology flanking the initiating double-stranded break to align the ends for repair

Note 1 to entry: MMEJ repair of DNA breaks in genome editing (1.2) approaches can result in deletion between pairs of microhomology regions.

Note 2 to entry: For DNA end-joining repair, the definition of the U.S. National Library of Medicine has been used.

3.6

non-homologous end joining

NHEJ

mechanism of DNA end-joining repair in which DNA ends are joined in a homology-independent manner

Note 1 to entry: NHEJ repair of DNA breaks in genome editing (1.2) workflows can result in indel (3.3) formation.

Note 2 to entry: For DNA end-joining repair, the definition of the U.S. National Library of Medicine has been used [3].

3.7

unintended edit

DNA, RNA, or epigenome intended edit

change to the genomic DNA sequence, RNA sequence, or epigenetic signature at an off-target (1.4) resulting from the application of genome editing (1.2) components (e.g. nuclease, repair template (2.1.1))

Note 1 to entry: See off-target (1.4), and edit (3.1)

Symbols and abbreviated terms

bp – base pairs

DNA – deoxyribonucleic acid

CRISPR – clustered regularly interspaced short palindromic repeats

RNA – ribonucleic acid

TAL – Transcription Activator-like

TALE – transcription activator-like effector

REFERENCES

  1. U.S. National Library of Medicine. MeSH Descriptor Data 2020: Transcription Activator-Like Effectors. https://meshb.nlm.nih.gov/record/ui?name=TRANSCRIPTION%20ACTIVATOR-LIKE%20EFFECTORS
  2. U.S. National Library of Medicine. MeSH Descriptor Data 2020: Recombinational DNA Repair. https://meshb.nlm.nih.gov/record/ui?ui=D059767
  3. U.S. National Library of Medicine. MeSH Descriptor Data 2020: DNA End-Joining Repair. https://meshb.nlm.nih.gov/record/ui?ui=D059766
Created November 18, 2020, Updated November 20, 2020