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.
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 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.
The restriction endonuclease Fok1, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non sequence-specific DNA cleavage domain at the C-terminal. Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3' recognition site, the DNA cleavage domain is activated and cleaves the DNA at two locations, regardless of the nucleotide sequence at the cut site. The DNA is cut 9 nucleotides downstream of the motif on the forward strand, and 13 nucleotides downstream of the motif on the reverse strand, producing two sticky ends with 4-bp overhangs.
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.
No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.
NgAgo is a single-stranded DNA (ssDNA)-guided Argonaute endonuclease, an acronym for NatronobacteriumgregoryiArgonaute. NgAgo binds 5′ phosphorylated ssDNA of ~24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. Like the CRISPR/Cas system, NgAgo was reported by Chunyu Han et al. to be suitable for genome editing, but this has not been replicated. In contrast to Cas9, the NgAgo–gDNA system does not require a protospacer adjacent motif (PAM).
CRISPR activation (CRISPRa) is a type of CRISPR tool that uses modified versions of CRISPR effectors without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs).
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.
CRISPR gene editing 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.
Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.
Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations. The approach utilises the CRISPR-Cas9 gene editing system, coupled with libraries of single guide RNAs (sgRNAs), which are designed to target every gene in the genome. Over recent years, the genome-wide CRISPR screen has emerged as a powerful tool for performing large-scale loss-of-function screens, with low noise, high knockout efficiency and minimal off-target effects.
CRISPR RNA or crRNA is a RNA transcript from the CRISPR locus. CRISPR-Cas is an adaptive immune system found in bacteria and archaea to protect against mobile genetic elements, like viruses, plasmids, and transposons. The CRISPR locus contains a series of repeats interspaced with unique spacers. These unique spacers can be acquired from MGEs.
The Fanzor (Fz) protein is an eukaryotic, RNA-guided DNA endonuclease, which means it is a type of DNA cutting enzyme that uses RNA to target genes of interest. It has been recently discovered and explored in a number of studies. In bacteria, RNA-guided DNA endonuclease systems, such as the CRISPR/Cas system, serve as an immune system to prevent infection by cutting viral genetic material. Currently, CRISPR/Cas9-mediated’s DNA cleavage has extensive application in biological research, and wide-reaching medical potential in human gene editing.
