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Gene Knocking-down
This technique allows the reduction of the expression of one or more of an organism. This can occur 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.
If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this leads to a temporary change in gene expression that does not modify the chromosomal DNA, and the result is referred to as a "transient knockdown".
In a transient knockdown, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression. Binding can occur through the blocking of transcription, the degradation of the mRNA transcript (e.g. siRNA or RNase-H dependent antisense), or through the blocking of either mRNA translation, pre-mRNA splicing sites, or nuclease cleavage sites used for maturation of other functional RNAs, including miRNA (e.g. by morpholino oligos).
The most direct use of transient knockdowns is for learning about a gene that has been sequenced, but has an unknown function. This experimental approach is known as reverse genetics. Transient knockdowns are often used in developmental biology because oligos can be injected into single-celled zygotes and will be present in the daughter cells of the injected cell.

RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation. Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Once introduced into the cell, exogenous siRNAs are processed by RISC. The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease.
RNAi is used for genetic functional analysis. The use of RNAi may be useful in identifying potential therapeutic targets, drug development, or other applications.

Gene Knocking-out
Gene knockout (KO) is a genetic technique in which one of an organism's genes is made inoperative. Knockout organisms are used to study gene function, usually by investigating the effect of gene loss. Heterozygous and homozygous Kos: in the former, only one allele is knocked out, in the latter both the two alleles are knocked out.

The directed creation of a KO begins in the test tube with a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell culture. Cells are transfected with the DNA construct. Often the goal is to create a transgenic animal that has the altered gene.
If so, embryonic stem cells are genetically transformed and inserted into early embryos. Resulting animals with the genetic change in their germline cells can then often pass the gene knockout to future generations.

The construct is engineered to recombine with the target gene, which is done by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene.
A conditional knockout allows gene deletion in a tissue or time specific manner. This is done by introducing loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-out. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites.
Because the DNA recombination is a rare event, the foreign sequence chosen for insertion usually includes a reporter. This enables easy selection of cells or individuals in which knockout was successful. Sometimes the DNA construct inserts into a chromosome without the desired homologous recombination with the target gene.

 (Ivana Venezia)

KNOCKDOWN
It is a technique by which the expression of a gene is reduced. This reduction can result from a DNA modification or from an oligonucleotide binding either to the mRNA or to the gene. In this case the expression change is temporary, so we talk about transient knockdown.
One of the techniques allowing the transient knockdown of a gene consists in the use of Morpholino oligomers. They have become a standard knockdown tool in animal embryonic systems, so Morpholino oligos are often used to investigate the role of a specific mRNA transcript in an embryo. Its molecular structure has DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos block access of other molecules to small (~25 base) specific sequences of the base-pairing surfaces of ribonucleic acid (RNA). Because of their completely unnatural backbones, Morpholinos are not recognized by cellular proteins. Nucleases do not degrade Morpholinos, nor are they degraded in serum or in cells. There are no published reports of Morpholinos activating toll-like receptors or innate immune responses such as interferon induction or the NF-κB-mediated inflammation response. Morpholinos are not known to modify DNA methylation.
Morpholinos do not trigger the degradation of their target RNA molecules, unlike many antisense structural types (e.g., phosphorothioates, siRNA). Instead, Morpholinos act by "steric blocking", binding to a target sequence within an RNA, inhibiting molecules that might otherwise interact with the RNA.
Morpholinos can also modify the splicing of pre-mRNA or inhibit the maturation and activity of miRNA.
Blocking translation
Bound to the 5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon. This prevents translation of the coding region of the targeted transcript (called "knocking down" gene expression). This is useful experimentally when an investigator wishes to know the function of a particular protein; Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism.
Modifying pre-mRNA splicing
Morpholinos can interfere with pre-mRNA processing steps either by preventing splice-directing small nuclear ribonucleoproteins (snRNP) complexes from binding to their targets at the borders of introns on a strand of pre-mRNA, or by blocking the nucleophilic adenine base and preventing it from forming the splice lariat structure, or by interfering with the binding of splice regulatory proteins such as splice silencers and splice enhancers. Preventing the binding of snRNP U1 (at the donor site) or U2/U5 (at the polypyrimidine moiety and acceptor site) can cause modified splicing, commonly excluding exons from the mature mRNA. Targeting some splice targets results in intron inclusions, while activation of cryptic splice sites can lead to partial inclusions or exclusions. Targets of U11/U12 snRNPs can also be blocked. Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction (RT-PCR) and is seen as a band shift after gel electrophoresis of RT-PCR products.
KNOCKOUT
A variation of the traditional gene knockout is the Conditional gene knockout. 
Conditional gene knockout is a technique used to eliminate a specific gene in a certain tissue, such as the liver. This technique is useful to study the role of individual genes in living organisms. It differs from traditional gene knockout because it targets specific genes at specific times rather than being deleted from beginning of life. Using the conditional gene knockout technique eliminates many of the side effects from traditional gene knockout. In traditional gene knockout, embryonic death from a gene mutation can occur, and this prevents scientists from studying the gene in adults. Some tissues cannot be studied properly in isolation, so the gene must be inactive in a certain tissue while remaining active in others. With this technology, scientists are able to knockout genes at a specific stage in development and study how the knockout of a gene in one tissue affects the same gene in other tissues.
The most commonly used technique is the Cre-lox recombination system. The Cre recombinase enzyme specifically recognizes two lox (loci of recombination) sites within DNA and causes recombination between them. This recombination will cause a deletion or inversion of the genes between the two lox sites, depending on their orientation. An entire gene can be removed or inactivated. This whole system is inducible so a chemical can be added to knock genes out at a specific time. Two of the most commonly used chemicals are tetracycline, which activates transcription of the Cre recombinase gene and tamoxifen, which activates transport of the Cre recombinase protein to the nucleus. Only a few cell types express Cre recombinase and no mammalian cells express it so there is no risk of accidental activation of lox sites when using conditional gene knockout in mammals.
A mouse containing the Cre gene and a mouse containing the loxP sequences are bred to generate a conditional knockout for a particular gene of interest. The mice do not naturally express Cre recombinase or lox sites but they have been engineered to express these gene products to create the desirable offspring. You have to obtain a mouse in which an important exon is floxed (it means that it has a Lox-P upstream and downstream) and a mouse which has a Cre sequence whose expression is regulated by a cell type specific promoter or an inducible promoter. Then you cross these mice and you obtain a mouse in which the cells non expressing Cre will have the gene with a normal function, while the cells expressing Cre will have a gene function disrupted in the portion between Lox-P.

(Francesca Luca)

RNA interferrence mechanism 

The RNA interference (RNAi), an ancient cellular antiviral response, is a natural mechanism for silencing gene expression. It can be exploited to allow specific inhibition of function of any target gene. RNAi is proving to be an invaluable research tool, it aids in the identification of novel genes involved in disease processes.

RNAi is not the only mechanism of silencing gene expression (table 1).

Table 1: Comparison between different methods for gene silencing.

 

RNA interference (RNAi) occurs in response to the introduction of double-stranded RNA (dsRNA) into a cell. The dsRna introduction triggers the activation of dsRNA-dependent protein kinase-R (PKR). Activated PKR phosphorylates the translation initiation factor EIF2: this effect, in association with activation of Rnase-L and induction of interferon production, halts protein synthesis and promotes apoptosis. Overall, this is believed to represent an antiviral defense mechanism. RNAi is a highly conserved mechanism throughout taxonomical species . In addition to have an antiviral activity, RNAi is also believed to suppress the expression of potentially harmful segments of the genome, such as transposons, which might otherwise destabilize the genome by acting as insertional mutagens.

Though its mechanisms are not fully elucidated, RNAi represents the result of a multistep process (Figure 1). Upon entering the cell, long dsRNAs are first processed by the RNAse III enzyme Dicer. This functional dimer contains helicase, dsRNA binding, and PAZ domains (their roles are not completely elucidated). Dicer produces 21–23 nucleotide dsRNA fragments with two nucleotide 3' end overhangs, i.e. siRNAs. RNAi is mediated by the RNA-induced silencing complex (RISC) which, guided by siRNA, recognizes mRNA containing a sequence homologous to the siRNA and cleaves the mRNA at a site located approximately in the middle of the homologous region. Thus, gene expression is specifically inactivated at a post-transcriptional level.

In C. elegans, Dicer has been shown to interact with rde proteins. The rde proteins bind to long dsRNA and are believed to present the long dsRNA to Dicer for processing. Mutants displaying a high degree of resistance to RNAi have been reported to possess mutations at rde-1 and rde-4 loci.

Figure 1: Mechanism of RNA interference

 The appearance of double stranded (ds) RNA within a cell (e.g. as a consequence of viral infection) triggers a complex response, which includes among other phenomena (e.g. interferon production and its consequences) a cascade of molecular events known as RNAi. During RNAi, the cellular enzyme Dicer binds to the dsRNA and cleaves it into short pieces of ~ 20 nucleotide pairs in length known as small interfering RNA (siRNA). These RNA pairs bind to the cellular enzyme called RNA-induced silencing complex (RISC) that uses one strand of the siRNA to bind to single stranded RNA molecules (i.e. mRNA) of complementary sequence. The nuclease activity of RISC then degrades the mRNA, thus silencing expression of the viral gene. Similarly, the genetic machinery of cells is believed to utilize RNAi to control the expression of endogenous mRNA, thus adding a new layer of post-transciptional regulation. RNAi can be exploited in the experimental settings to knock down target genes of interest with a high specific and relatively easy technology (see text for more details).

 Besides gene silencing, RNAi might be involved in other phenomena of gene regulation.. It appears that RNAi can also function by methylating cytosines as well as CpG sequences more classically associated with methylation. If the target sequence shares homology with a promoter, transcriptional silencing may occur via methylation. Moreover, RNA appears to interact with chromatin domains, which may ultimately direct DNA methylation. Studies of C. elegans have shown that RNAi can spread among cells through mechanisms that may not hinge upon siRNA. The systemic RNA interference-deficient (sid) locus, sid-1, encodes a conserved protein with a signal peptide sequence and 11 putative transmembrane domains, suggesting that the sid-1 protein may act as a channel for long dsRNA, siRNA, or a currently undiscovered RNAi-related signal. Sid-1 mutants retain cell-autonomous RNAi but fail to show spreading of RNAi. It remains unclear whether this systemic RNAi occurs in mammals, although a strong similarity is reported between sid-1 and predicted human and mouse proteins.

(Justine Floret)

Source : https://translational-medicine.biomedcentral.com/articles/10.1186/1479-5876-2-39#Sec3

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ZFN, TALEN , CRIPR/CAS9

These three molecular technologies allow to edite genome in a site specific manner, each technique with a different mechanism. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)  are chimeric nucleases composed of programmable, sequence-specific DNA-binding modules linked to a non-specific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone non-homologous end joining or homology-directed repair at specific genomic locations.

Image taken from http://gentaur-italy.com/prodotti/talen-un-sistema-semplice-e-veloce-per-gene-knockout/

The versatility of ZFNs and TALENs arises from the ability to customize the DNA-binding domain to recognize virtually any sequence. These DNA-binding modules can be combined with numerous effector domains to impact genomic structure and function, including nucleases, transcriptional activators and repressors, recombinases, transposases, DNA histone methyltransferases and histone acetyltransferases. Thus, the ability to successfully execute genetic alterations depends largely on the DNA-binding specificity and affinity of designed zinc-finger and TALE proteins.

Cys2-His2 zinc finger proteins 

The Cys₂-His₂ zinc-finger domain is among the most common types of DNA-binding motifs found in eukaryotes and represents the second most frequently encoded protein domain in the human genome. An individual zinc-finger consists of approximately 30 amino acids in a conserved ββα configuration. Several amino acids on the surface of the α-helix typically contact three base pairs in the major groove of DNA, with varying levels of selectivity. The modular structure of zinc-finger proteins has made them an attractive framework for the design of custom DNA-binding proteins. Key to the application of zinc-finger proteins for specific DNA recognition was the development of unnatural arrays that contain more than three zinc-finger domains. This advance was facilitated by the structure-based discovery of a highly conserved linker sequence that enabled construction of synthetic zinc-finger proteins that recognized DNA sequences 9 to 18 bps in length. Because 18 bps of DNA sequence can confer specificity within 68 billion bp of DNA, this method allowed for specific sequences to be targeted in the human genome for the first time.

Transcription Activator-like effectors

TALEs are naturally occurring proteins from the plant pathogenic bacteria genus Xanthomonas, and contain DNA-binding domains composed of a series of 33–35 amino acid repeat domains that each recognizes a single bp. TALE specificity is determined by two hypervariable amino acids that are known as the repeat-variable diresidues (RVDs) . Like zinc-fingers, modular TALE repeats are linked together to recognize contiguous DNA sequences. However in contrast to zinc finger proteins, there was no re-engineering of the linkage between repeats necessary to construct long arrays of TALEs with the ability to theoretically address single sites in the genome. In addition to their role in facilitating HDR (Homologous direct recombination), site-specific nucleases also allow rapid generation of cell lines and organisms with null phenotypes; NHEJ-mediated repair of a nuclease-induced DSB leads to the introduction of small insertions or deletions at the targeted site, resulting in knockout of gene function via frame-shift mutations.

Image taken from http://www.anim.med.kyoto-u.ac.jp/gma/nucleases.aspx

Distinct from the site-specific nucleases described above, the CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/CRISPR-associated (Cas) system has recently emerged as a potentially facile and efficient alternative to ZFNs and TALENs for inducing targeted genetic alterations. In bacteria, the CRISPR system provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage. In the Type II CRISPR/Cas system, short segments of foreign DNA, termed “spacers” are integrated within the CRISPR genomic loci and transcribed and processed into short CRISPR RNA (crRNAs). These crRNAs anneal to trans-activating crRNAs (tracrRNAs) and direct sequence-specific cleavage and silencing of pathogenic DNA by Cas proteins. Recent work has shown that target recognition by the Cas9 protein requires a “seed” sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA- binding region. The CRISPR/Cas system can thereby be re-targeted to cleave virtually any DNA sequence by re-designing the crRNA. Significantly, the CRISPR/Cas system has been shown to be directly portable to human cells by co-delivery of plasmids expressing the Cas9 endonuclease and the necessary crRNA components.

Image taken from https://www.computescotland.com/crisprcas9-genome-editing-8111.php

Source

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694601/