A restriction enzyme, restriction endonuclease, REase, ENase orrestrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.
A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) which stabilizes 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.
Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.
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.
EcoRV is a type II restriction endonuclease isolated from certain strains of Escherichia coli. It has the alternative name Eco32I.
In Molecular biology, an insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism.
Recombinant adeno-associated virus (rAAV) based genome engineering is a genome editing platform centered on the use of recombinant AAV 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. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).
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.
Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.
CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna. Sequence-specific activation of gene expression refers to CRISPR activation (CRISPRa).
Epigenome editing or epigenome engineering is a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites. Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.
A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.
Cas12a is a subtype of Cas12 proteins and an RNA-guided endonuclease that forms part of the CRISPR system in some bacteria and archaea. It originates as part of a bacterial immune mechanism, where it serves to destroy the genetic material of viruses and thus protect the cell and colony from viral infection. Cas12a and other CRISPR associated endonucleases use an RNA to target nucleic acid in a specific and programmable matter. In the organisms from which it originates, this guide RNA is a copy of a piece of foreign nucleic acid that previously infected the cell.
EcoRI is a restriction endonuclease enzyme isolated from species E. coli. It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modification system. The Eco part of the enzyme's name originates from the species from which it was isolated - "E" denotes generic name which is "Escherichia" and "co" denotes species name, "coli" - while the R represents the particular strain, in this case RY13, and the I denotes that it was the first enzyme isolated from this strain.
J. Keith Joung is an American pathologist and molecular biologist who holds the Robert B. Colvin Endowed Chair in Pathology at Massachusetts General Hospital and is Professor of Pathology at Harvard Medical School. He is a leading figure in the field of genome editing and has pioneered the development of designer nucleases and sensitive off-target detection methods.
Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave, creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.
Since antiretroviral therapy requires a lifelong treatment regimen, research to find more permanent cures for HIV infection is currently underway. It is possible to synthesize zinc finger nucleotides with zinc finger components that selectively bind to specific portions of DNA. Conceptually, targeting and editing could focus on host cellular co-receptors for HIV or on proviral HIV DNA.
