Deoxyribonucleic acid is a polymer composed of two polynucleotide chains that coil around each other to form a double helix carrying genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.
In molecular biology, a transcription factor (TF) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The function of TFs is to regulate—turn on and off—genes in order to make sure that they are expressed in the desired cells at the right time and in the right amount throughout the life of the cell and the organism. Groups of TFs function in a coordinated fashion to direct cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and intermittently in response to signals from outside the cell, such as a hormone. There are up to 1600 TFs in the human genome. Transcription factors are members of the proteome as well as regulome.
A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) in order to stabilize the fold. It was originally coined to describe the finger-like appearance of a hypothesized structure from the African clawed frog (Xenopus laevis) transcription factor IIIA. However, it has been found to encompass a wide variety of differing protein structures in eukaryotic cells. Xenopus laevis TFIIIA was originally demonstrated to contain zinc and require the metal for function in 1983, the first such reported zinc requirement for a gene regulatory protein followed soon thereafter by the Krüppel factor in Drosophila. It often appears as a metal-binding domain in multi-domain proteins.
DNA-binding proteins are proteins that have DNA-binding domains and thus have a specific or general affinity for single- or double-stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, because it exposes more functional groups that identify a base pair. However, there are some known minor groove DNA-binding ligands such as netropsin, distamycin, Hoechst 33258, pentamidine, DAPI and others.
CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes and provide a form of acquired immunity. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence or have a general affinity to DNA. Some DNA-binding domains may also include nucleic acids in their folded structure.
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Alongside CRISPR/Cas9 and TALEN, ZFN is a prominent tool in the field of genome editing.
The gene-for-gene relationship was discovered by Harold Henry Flor who was working with rust of flax. Flor showed that the inheritance of both resistance in the host and parasite ability to cause disease is controlled by pairs of matching genes. One is a plant gene called the resistance (R) gene. The other is a parasite gene called the avirulence (Avr) gene. Plants producing a specific R gene product are resistant towards a pathogen that produces the corresponding Avr gene product. Gene-for-gene relationships are a widespread and very important aspect of plant disease resistance. Another example can be seen with Lactuca serriola versus Bremia lactucae.
Type three secretion system is a protein appendage found in several Gram-negative bacteria.
Gene targeting is a genetic technique that uses homologous recombination to modify an endogenous gene. The method can be used to delete a gene, remove exons, add a gene and modify individual base pairs. The process of gene targeting provides a way to alter specific genes in order to better identify their biological roles. Gene targeting can be permanent or conditional. Conditions can be a specific time during development / life of the organism or limitation to a specific tissue, for example. Gene targeting requires the creation of a specific vector for each gene of interest. However, it can be used for any gene, regardless of transcriptional activity or gene size.
CHEK2 is a tumor suppressor gene that encodes the protein CHK2, a serine-threonine kinase. CHK2 is involved in DNA repair, cell cycle arrest or apoptosis in response to DNA damage. Mutations to the CHEK2 gene have been linked to a wide range of cancers.
Ski-like protein is a protein that in humans is encoded by the SKIL gene.
Zinc finger protein chimera are chimeric proteins composed of a DNA-binding zinc finger protein domain and another domain through which the protein exerts its effect. The effector domain may be a transcriptional activator (A) or repressor (R), a methylation domain (M) or a nuclease (N).
TALeffectors are proteins secreted by some β- and γ-proteobacteria. Most of these are Xanthomonads. Plant pathogenic Xanthomonas bacteria are especially known for TALEs, produced via their type III secretion system. These proteins can bind promoter sequences in the host plant and activate the expression of plant genes that aid bacterial infection. The TALE domain responsible for binding to DNA is known to have 1.5 to 33.5 short sequences that are repeated multiple times. Each of these repeats was found to be specific for a certain base pair of the DNA. These repeats also have repeat variable residues (RVD) that can detect specific DNA base pairs. They recognize plant DNA sequences through a central repeat domain consisting of a variable number of ~34 amino acid repeats. There appears to be a one-to-one correspondence between the identity of two critical amino acids in each repeat and each DNA base in the target sequence. These proteins are interesting to researchers both for their role in disease of important crop species and the relative ease of retargeting them to bind new DNA sequences. Similar proteins can be found in the pathogenic bacterium Ralstonia solanacearum and Burkholderia rhizoxinica, as well as yet unidentified marine microorganisms. The term TALE-likes is used to refer to the putative protein family encompassing the TALEs and these related proteins.
Recombinant adeno-associated virus (rAAV) based genome engineering is a genome editing platform centered on the use of recombinant rAAV vectors that enables insertion, deletion or substitution of DNA sequences into the genomes of live mammalian cells. The technique builds on Mario Capecchi and Oliver Smithies' Nobel Prize–winning discovery that homologous recombination (HR), a natural hi-fidelity DNA repair mechanism, can be harnessed to perform precise genome alterations in mice. rAAV mediated genome-editing improves the efficiency of this technique to permit genome engineering in any pre-established and differentiated human cell line, which, in contrast to mouse ES cells, have low rates of HR.
Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.
Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site specific locations.
Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.
Transcription Activator-Like Effector-Likes (TALE-likes) are a group of bacterial DNA binding proteins named for the first and still best-studied group, the TALEs of Xanthomonas bacteria. TALEs are important factors in the plant diseases caused by Xanthomonas bacteria, but are known primarily for their role in biotechnology as programmable DNA binding proteins, particularly in the context of TALE nucleases. TALE-likes have additionally been found in many strains of the Ralstonia solanacearum bacterial species complex, in Paraburkholderia rhizoxinica strain HKI 454, and in two unknown marine bacteria. Whether or not all these proteins form a single phylogenetic grouping is as yet unclear.
Daniel Voytas, Ph.D., is Professor of Genetics, Cell Biology and Development at the University of Minnesota and Director of the Center for Precision Plant Genomics. He is also the Chief Scientific Officer of Calyxt, an agricultural biotechnology company focused on developing crops that provide consumer benefit.
