Molecular biology is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions.
Molecular genetics is a branch of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens.
In genetics, an insertion is the addition of one or more nucleotide base pairs into a DNA sequence. This can often happen in microsatellite regions due to the DNA polymerase slipping. Insertions can be anywhere in size from one base pair incorrectly inserted into a DNA sequence to a section of one chromosome inserted into another. The mechanism of the smallest single base insertion mutations is believed to be through base-pair separation between the template and primer strands followed by non-neighbor base stacking, which can occur locally within the DNA polymerase active site. On a chromosome level, an insertion refers to the insertion of a larger sequence into a chromosome. This can happen due to unequal crossover during meiosis.
M13 is one of the Ff phages, a member of the family filamentous bacteriophage (inovirus). Ff phages are composed of circular single-stranded DNA (ssDNA), which in the case of the m13 phage is 6407 nucleotides long and is encapsulated in approximately 2700 copies of the major coat protein p8, and capped with about 5 copies each of four different minor coat proteins. The minor coat protein p3 attaches to the receptor at the tip of the F pilus of the host Escherichia coli. The life cycle is relatively short, with the early phage progeny exiting the cell ten minutes after infection. Ff phages are chronic phage, releasing their progeny without killing the host cells. The infection causes turbid plaques in E. coli lawns, of intermediate opacity in comparison to regular lysis plaques. However, a decrease in the rate of cell growth is seen in the infected cells. The replicative form of M13 is circular double-stranded DNA similar to plasmids that are used for many recombinant DNA processes, and the virus has also been used for phage display, directed evolution, nanostructures and nanotechnology applications.
CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. Each sequence within an individual prokaryotic cell is derived from a DNA fragment of a bacteriophage that had previously infected the prokaryote or one of its ancestors. These sequences 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 heritable, acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.
Guide RNA (gRNA) or single guide RNA (sgRNA) is a short sequence of RNA that functions as a guide for the Cas9-endonuclease or other Cas-proteins that cut the double-stranded DNA and thereby can be used for gene editing. In bacteria and archaea, gRNAs are a part of the CRISPR-Cas system that serves as an adaptive immune defense that protects the organism from viruses. Here the short gRNAs serve as detectors of foreign DNA and direct the Cas-enzymes that degrades the foreign nucleic acid.
In molecular cloning and biology, a gene knock-in refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found within the locus. Typically, this is done in mice since the technology for this process is more refined and there is a high degree of shared sequence complexity between mice and humans. The difference between knock-in technology and traditional transgenic techniques is that a knock-in involves a gene inserted into a specific locus, and is thus a "targeted" insertion. It is the opposite of gene knockout.
Daisy chaining DNA is a form of gene editing, or "gene drive", which, unlike CRISPR, is self-limiting. This means that any alteration made in the laboratory to a gene sequence is limited to a local population only, and cannot be passed on to global populations. It occurs when DNA undergoing PCR amplification forms tangles that resemble a 'daisy chain.' In essence it teaches DNA to count, so that the new strain will only reproduce for a fixed number of generations. It may be useful for instance to alter a particular strain of wheat to suit a local area with no danger of the new strain escaping into wild populations. As a new technique, it must be studied under carefully controlled conditions until it is better understood. Research is typically performed in closed systems on organisms such as yeast, fruit flies, mosquitos, and rapidly evolving nematode worms.
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).
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.
David Ruchien Liu is an American molecular biologist and chemist. He is Richard Merkin Professor, Director of the Merkin Institute of Transformative Technologies in Healthcare, and Vice-Chair of the Faculty at the Broad Institute of Harvard University and the Massachusetts Institute of Technology.
A gene drive is a natural process and technology of genetic engineering that propagates a particular suite of genes throughout a population by altering the probability that a specific allele will be transmitted to offspring. Gene drives can arise through a variety of mechanisms. They have been proposed to provide an effective means of genetically modifying specific populations and entire species.
Cas12a is a subtype of Cas12 proteins and an RNA-guided endonuclease that forms part of the CRISPR system in some bacteria and archaea. In CRISPR systems, Cas12a serves to destroy the genetic material of viruses and other foreign DNA, thereby protecting the cell from infection. Like other Cas enzymes, Cas12a binds to an RNA to target nucleic acid in a specific and programmable matter. In the host organism, the crRNA contains a constant region that is recognized by the Cas12a protein and a spacer region that is complementary to a piece of foreign nucleic acid that previously infected the cell.
CRISPR gene editing (CRISPR, pronounced "crisper", refers to "clustered regularly interspaced short palindromic repeats") is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.
Anti-CRISPR is a group of proteins found in phages, that inhibit the normal activity of CRISPR-Cas, the immune system of certain bacteria. CRISPR consists of genomic sequences that can be found in prokaryotic organisms, that come from bacteriophages that infected the bacteria beforehand, and are used to defend the cell from further viral attacks. Anti-CRISPR results from an evolutionary process occurred in phages in order to avoid having their genomes destroyed by the prokaryotic cells that they will infect.
Phage-assisted continuous evolution (PACE) is a phage-based technique for the automated directed evolution of proteins. It relies on relating the desired activity of a target protein with the fitness of an infectious bacteriophage which carries the protein's corresponding gene. Proteins with greater desired activity hence confer greater infectivity to their carrier phage. More infectious phage propagate more effectively, selecting for advantageous mutations. Genetic variation is generated using error-prone polymerases on the phage vectors, and over time the protein accumulates beneficial mutations. This technique is notable for performing hundreds of rounds of selection with minimal human intervention.
The eradication or abolition of suffering is the concept of using biotechnology to create a permanent absence of involuntary pain and suffering in all sentient beings.
Diversity-generating retroelements (DGRs) are a family of retroelements that were first found in Bordetella phage (BPP-1), and since been found in bacteria, Archaea, Archaean viruses, temperate phages, and lytic phages. DGRs benefit their host by mutating particular regions of specific target proteins, for instance, phage tail fiber in BPP-1, lipoprotein in legionella pneumophila, and TvpA in Treponema denticola . An error-prone reverse transcriptase is responsible for generating these hypervariable regions in target proteins. In mutagenic retrohoming, a mutagenized cDNA is reverse transcribed from a template region (TR), and is replaced with a segment similar to the template region called variable region (VR). Accessory variability determinant (Avd) protein is another component of DGRs, and its complex formation with the error-prone RT is of importance to mutagenic rehoming.
Make People Better is a 2022 documentary film about the use of genetic engineering to enhance two twins girls to be immune to HIV. Directed by Cody Sheehy of Rhumbline Media, it was originated by Samira Kiani, a biotechnologist then at Arizona State University. It focuses on the circumstances involving Chinese biologist He Jiankui who created the first genetically modified humans in 2018. Featured experts included Antonio Regalado, senior editor for biomedicine of MIT Technology Review, who first discovered and revealed the secret experiment, and Benjamin Hurlbut, a bioethicist at the Arizona State University.
The Innovative Genomics Institute (IGI) is an American nonprofit scientific research institute founded by Nobel laureate and CRISPR gene editing pioneer Jennifer Doudna and biophysicist Jonathan Weissman. The institute is based at the University of California, Berkeley, and also has member researchers at the University of California, San Francisco, UC Davis, UCLA, Lawrence Berkeley National Laboratory, Lawrence Livermore National Laboratory, Gladstone Institutes, and other collaborating research institutions. The IGI focuses on developing real-world applications of genome editing to address problems in human health, agriculture and climate change.
