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Difference between micro RNA and short-interfering RNA and CRISPR Cas 9 system?

Difference between micro RNA and short-interfering RNA and CRISPR Cas 9 system?


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I read this article https://www.quantamagazine.org/20150206-crispr-dna-editor-bacteria/ and am slightly puzzled as to why the CRISPR/Cas 9 system is seen as being so revolutionary. It seems like the very same thing that micro RNA and short interfering RNA does- cleaves synthesised mRNA strands by attaching to the complementary part of the mmRNA strand, and the restrictive enzymes combine to this RNA and cleave it. I don't see how the CRISPR system is any different or better…

Thank you in advance :)

EDIT: Also, does anyone know whether Cas 9 cuts both DNA strands at the same place, or whether it leaves 'sticky ends'? Thank you.


The CRISPR Cas 9 system is used to introduce insertion or deletion in a genomic sequence not mRNA.

https://www.addgene.org/CRISPR/guide/


Abstract

Clustered short palindrome repeats with regular intervals, abbreviated as CRISPR, and functions as a self-defense system for prokaryotes, detecting particular pathogenic nucleic acid, interfering with the functions of exoteric DNA, and protecting them against foreign invaders. In recent years, CRISPR has attracted increasing interests in the in vitro diagnostic field because of its inherent allele specificity, which is one of the critical factors for the successful application of this technology in the development of high-precision treatment and diagnosis. Herein, this review article aims to provide an overview of CRISPR-CRISPR associated proteins (Cas) based biomedical diagnostics, including the biological mechanism, biomaterials, and applications. This paper first briefly introduces the development history and biological characteristics of the CRISPR-Cas system, and then summarizes the application status and development trend of the CRISPR-Cas system in the detection and identification of particular pathogens, specifically displaying a brilliant prospect in the most recent outbreak of novel coronavirus (formerly named 2019-nCoV). Moreover, its potential diagnostic power in oncogene mutations and single nucleotide variations detecting are assembled. Finally, we discuss challenges and future prospects of CRISPR-Cas system based diagnostic platforms in biomedicine, hoping to further inspire the development of biomedical diagnostics.


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Contents

Some of the earliest asRNAs were discovered while investigating functional proteins. An example was micF asRNA. While characterizing the outer membrane porin ompC in E.coli, some of the ompC promoter clones observed were capable of repressing the expression of other membrane porin such as ompF. The region responsible for this repression function was found to be a 300 base-pair locus upstream of the ompC promoter. This 300 base-pair region is 70% homologous in sequence with the 5' end of the ompF mRNA and thus the transcript of this 300 base pair locus was complementary to the ompF mRNA. Later on, this transcript, denoted micF, was found to be an asRNA of ompF and capable of downregulating the expression of ompF under stress by forming a duplex with the ompF mRNA. This induces the degradation of the ompF mRNA. [2]

Unlike micF RNA being discovered by accident, the majority of asRNAs were discovered by genome wide searches for small regulatory RNAs and by transcriptome analysis. Conventionally, the first step involves computational predictions based on some known characteristics of asRNAs. During computational searches, the encoding regions are excluded. The regions that are predicted to have conserved RNA structures and act as orphan promoters and Rho independent terminators are preferenced during analysis. Because computational searches focuses on the intergenic region, the asRNAs that are transcribed from the opposite strand of an encoding gene are likely to be missed using this method. To detect asRNA transcribed from the encoding region, oligonucleotide microarrays can be used. In this method, one or both strands of encoding genes can be used as probes. In addition to computational searches and microarrays, some asRNAs were discovered by sequencing cDNA clones as well as mapping promoter elements. [7] Although many findings from the approaches mentioned above gave rise to a lot of possible asRNAs, only few were proven to be actual asRNAs via further functional tests. To minimize the number of false positive results, new approaches from recent years have been focusing on strand-specific transcription, chromatin binding noncoding RNAs and single cell studies. [1]

The idea of asRNAs as drug targets started in 1978 when Zamecnik and Stephenson found an antisense oligonucleotide to the viral RNA of Rous scarcoma virus that was capable of inhibiting viral replication and protein synthesis. Since then, much effort has been devoted to developing asRNAs as drug candidates. In 1998, the first asRNA drug, fomivirsen, was approved by FDA. Fomivirsen, a 21 base-pair oligonucleotide, was developed to treat cytomegalovirus retinitis in patients with AIDS. It works by targeting the transcribed mRNA of the virus and consequently inhibiting replication of cytomegalovirus. Despite fomivirsen was discontinued in 2004 due to the loss of the market, it served as a successful and inspiring example of using asRNAs as drug targets or drug candidates. [5]

Another example of using an asRNA as a therapeutic agent is mipomersen, which was approved by FDA in 2013. Mipomersen was developed to manage the level of low-density lipoprotein cholesterol (LDL) in patients with homozygous familial hypercholesterolemia (HoFH), which is a rare autosomal dominant genetic condition. Because of the high level of total cholesterol (650–1000 mg/dL) and LDL receptor (above 600 mg/dL) in HoFH, patients with HoFH has a high risk for coronary heart disease. Because the protein apo-B-100 has been found to be required to produce very low-density lipoprotein (VLDL) and LDL, mipomersen complements with the mRNA of apo-B-100 and target it for RNAse H dependent degradation. Ultimately, mipomersen is able to reduce the level of LDL. [8]

The initial asRNAs discovered were in prokaryotes including plasmids, bacteriophage and bacteria. For example, in plasmid ColE1, the asRNA termed RNA I plays an important role in determining the plasmid copy number by controlling replication. The replication of ColE1 relies on the transcription of a primer RNA named RNA II. Once RNA II is transcribed, it hybridizes to its DNA template and later cleaved by RNase H. In the presence of the asRNA RNA I, RNA I and RNA II forms a duplex which introduces a conformational change of RNA II. Consequently, RNA II cannot hybridize with its DNA template which results in a low copy number of ColE1. In bacteriophage P22, the asRNA sar helps regulate between lytic and lysogenic cycle by control the expression of Ant. [9] Besides being expressed in prokaryotes, asRNAs were also discovered in plants. The most well described example of asRNA regulation in plants is on Flowering Locus C (FLC) gene. FLC gene in Arabidopsis thaliana encodes for a transcription factor that prevent expression of a range of genes that induce floral transition. In cold environment, the asRNA of FLC gene, denoted COOLAIR, is expressed and inhibits the expression of FLC via chromatin modification which consequently allows for flowering. [10] In mammalian cells, a typical example of asRNA regulation is X chromosome inactivation. Xist, an asRNA, can recruit polycomb repressive complex 2 (PRC2) which results in heterochromatinization of the X chromosome. [3]

Antisense RNAs can be classified in different ways. In terms of regulatory mechanisms, some author group asRNAs into RNA-DNA interactions, RNA-RNA interactions either in nucleus or cytoplasm and RNA-protein interactions (epigenetic). [3] Antisense RNAs can be categorized by the type of the promoters that initiate expression of asRNAs: independent promoters, shared bidirectional promoters or cryptic promoters. In terms of length, although asRNA in general is classified under lncRNAs, there are short asRNAs with length of less than 200 base pairs. Because the regulatory mechanism of asRNAs are found to be species specific, asRNAs can also be classified by species. [1] One of the common ways of classifying asRNAs is by where the asRNAs are transcribe relatively to their target genes: cis-acting and trans-acting.

Cis-acting Edit

Cis-acting asRNAs are transcribed from the opposite strand of the target gene at the target gene locus. They often show high degree or complete complementarity with the target gene. If the cis-acting asRNA regulates gene expression by targeting mRNA, it can only target individual mRNA. Upon interactions with the targeting mRNAs, cis-acting asRNAs can either block ribosome binding or recruit RNAase to degrade the targeting mRNAs. Consequently, the function of these cis-acting asRNAs is to repress translation of the targeting mRNAs. [2] Besides cis-acting asRNAs that target mRNAs, there are cis-acting epigenetic silencers and activators. Antisense RNA has been shown to repress the translation of LINE1-ORF2 domain of Entamoeba histolytica. However it is not confirmed yet whether its cis-acting or trans. [11]

In terms of epigenetic modification, cis-acting refers to the nature of these asRNAs that regulate epigenetic changes around the loci where they are transcribed. Instead of targeting individual mRNAs, these cis-acting epigenetic regulators can recruit chromatin modifying enzymes which can exert effects on both the transcription loci and the neighboring genes. [3]

Trans-acting Edit

Trans-acting asRNAs are transcribed from loci that are distal from the targeting genes. In contrast to cis-acting asRNAs, they display low degree of complementarity with the target gene but can be longer than cis-acting asRNAs. They can also target multiple loci. Because of these properties of trans-acting asRNAs, they form less stable complexes with their targeting transcripts and sometimes require aids from RNA chaperone protein such as Hfq to exert their functions. Due to the complexity of the trans-acting asRNAs, they are currently considered as less druggable targets. [2]

Function Edit

Epigenetic regulation Edit

Many examples of asRNAs show the inhibitory effect on transcription initiation via epigenetic modifications.

DNA methylation Edit

DNA methylation can result in long term downregulation of specific genes. Repression of functional proteins via asRNA induced DNA methylation has been found in several human disease. In a class of alpha-thalassemia, a type of blood disorder that has reduced level of hemoglobin leading to insufficient oxygen in the tissues, [12] hemoglobin alpha1 gene (HBA1) is downregulated by an abnormal transcript of putative RNA-binding protein Luc7-like (LUC71) that serves as an asRNA to HBA1 and induces methylation of HBA1's promoter. [1] Another example is silencing of a tumor suppressor gene p15INK4b, also called CDKN2B, in acute lymphoblastic leukemia and acute myeloid leukemia. The asRNA that is responsible for this silencing effect is antisense non-coding RNA in the INK locus (ANRIL), which is expressed in the same locus that encodes for p15INK4b. [3]

Histone modification Edit

In eukaryotic cells, DNA is tightly packed by histones. Modification on histones can change interactions with DNA which can further induce changes in gene expression. The biological consequences of histone methylation are context dependent. In general, histone methylation leads to gene repression but gene activation can also be achieved. [13] Evidence has shown histone methylation can be induced by asRNAs. For instance, ANRIL, in addition to the ability to induce DNA methylation, can also repress the neighboring gene of CDKN2B, CDKN2A, by recruiting polycomb repressive complex 2 (PRC2) which leads to histone methylation (H3K27me). Another classic example is X chromosome inactivation by XIST. [1]

ANRIL induced epigenetic modification is an example of cis acting epigenetic regulation. [3] In addition, Antisense RNA-induced chromatin modification can be both trans-acting. For example, in mammals, the asRNA HOTAIR is transcribed from homeobox C (HOXC) locus but it recruits PRC2 to HOXD which deposits H3K27 and silences HOXD. HOTAIR is highly expressed in primary breast tumors. [1]

Co-transcriptional regulation Edit

Epigenetic regulations such as DNA methylation and histone methylation can repress gene expression by inhibiting initiation of transcription. Sometimes, however, gene repression can be achieved by prematurely terminating or slowing down transcription process. AsRNAs can be involved in this level of gene regulation. For example, in bacterial or eukaryotic cells where complex RNA polymerases are present, bidirectional transcription at the same locus can lead to polymerase collision and results in the termination of transcription. Even when polymerase collision is unlikely during weak transcription, polymerase pausing can also occur which blocks elongation and leads to gene repression. One of the examples is repression of IME4 gene by its asRNA RME2. Another way of affecting transcription co-transcriptionally is by blocking splicing. One classic example in human is zinc-finger E-box binding homeobox 2 gene (ZEB2) which encodes E-cadherin, a transcriptional repressor. Efficient translation of ZEB2 mRNA requires the presence of an internal ribosome entry site (IRES) in intron of the mRNA at the 5' end. With the asRNA of ZEB2 being expressed, it can mask the splicing site and maintain the IRES in the mRNA which results in an efficient synthesis of E-cadherin. Lastly, depending on the level of asRNA expression, different isoforms of the sense transcript can be produced. Therefore, asRNA dependent regulation is not limited to on/off mechanism rather, it presents a fine tone control system. [1]

Post-transcriptional regulation Edit

The direct post transcriptional modulation by asRNAs refers to mRNAs being targeted by asRNAs directly thus, the translation is affected. Some characteristics of this type of asRNAs are described in the cis- and trans- acting asRNAs. This mechanism is relatively fast because both the targeting mRNA and its asRNA need to be present simultaneously in the same cell. As described in the cis-acting asRNAs, the mRNA-asRNA pairing can result in blockage of ribosome entry and RNase H dependent degradation. Overall, mRNA-targeting asRNAs can either activate or inhibit translation of the sense mRNAs with inhibitory effect being the most abundant. [1]

Therapeutic potential Edit

As a regulatory element, asRNAs bear many advantages to be considered as a drug target. First of all, asRNAs regulate gene expression at multiple levels including transcription, post-transcription and epigenetic modification. Secondly, the cis-acting asRNAs are sequence specific and exhibits high degree of complementarity with the targeting genes. [1] Thirdly, the expression level of asRNAs is very small compared to that of the targeting mRNAs therefore, only small amount of asRNAs is required to produce an effect. In terms of drug targets, this represents a huge advantage because only a low dosage is required for effectiveness. [4]

Recent years the idea of targeting asRNAs to increase gene expression in a locus specific manner has been drawing much attention. Due to the nature of drug development, it is always easier to have drugs functioning as downregulators or inhibitors. However, there is a need in developing drugs that can activate or upregulate gene expression such as tumor suppressor genes, neuroprotective growth factors and genes that are found silenced in certain Mendelian disorders. Currently, the approach to restore deficient gene expression or protein function include enzyme replacement therapies, microRNA therapies and delivery of functional cDNA. However, each bears some drawbacks. For example, the synthesized protein used in the enzyme replacement therapies often cannot mimic the whole function of the endogenous protein. In addition, enzyme replacement therapies are life-long commitment and carry a large financial burden for the patient. Because of the locus specific nature of asRNAs and evidences of changes in asRNA expression in many diseases, there have been attempts to design single stranded oligonucleotides, referred as antagoNATs, to inhibit asRNAs and ultimately to increase specific gene expression. [4]

Despite the promises of asRNAs as drug targets or drug candidates, there are some challenges remained to be addressed. [14] First of all, asRNAs and antagoNATs can be easily degraded by RNase or other degrading enzymes. To prevent degradation of the therapeutic oliogoneucleotides, chemical modification is usually required. The most common chemical modification on the oligonucleotides is adding a phosphorothioate linkage to the backbones. [5] However, the phosphrothioate modification can be proinflammatory. Adverse effects including fever, chills or nausea have been observed after local injection of phosphrothioate modified oligonucleotides. Secondly, off target toxicity also represents a big problem. Despite the locus-specific nature of the endogenous asRNAs, only 10–50% synthesized oligonucleotides showed expected targeting effect. One possible reason for this problem is the high requirement on the structure of the asRNAs to be recognized by the target sequence and RNase H. A single mismatch can result in distortion in the secondary structure and lead to off target effects. [4] Lastly, artificial asRNAs have been shown to have limited intracellular uptake. [5] Although neurons and glia have been shown to have the ability to freely uptake naked antisense oligonucleotides, a traceable carriers such as virus and lipid vesicles would still be ideal to control and monitor the intracellular concentration and metabolism. [4]


DISCUSSION

Although the M. tuberculosis CRISPR/Cas system has been widely exploited to analyze the evolution and epidemiology of this ancient pathogen, the work presented here represents the first in-depth functional characterization of this type III-A system. We have demonstrated that it has distinctive features that set it apart from other type III-A systems its mature crRNAs are of uniform length (∼71 nt), their structure (intact spacer flanked by 3′ and 5′ repeat sequence tags, the 3′ tag forming a hairpin structure) closely resembling that of type I system mature crRNAs. A single Cas6 cleavage event of repeat RNA 8 nt from its 3′ end appears to be sufficient to generate mature crRNA. M. tuberculosis Cas6 also has distinctive features: it is strongly promoted by metal ions, and, although the 3′ hairpin formed by the repeat sequence is important for accurate Cas6 cleavage, Cas6 RNA cleavage activity per se is independent of RNA secondary structure. These atypical features further exemplify the diversity that is found among CRISPR/Cas systems, even within the 1 type, and likely point to further diversity in functional mechanisms between systems. Such mechanistic variation between systems raises the possibility of designing novel type III system-based gene editing systems.

The key difference in the generation of type III and type I system crRNAs is that, although primary processing in both systems is completed by endoribonuclease Cas6, type III system crRNAs typically undergo further extensive 3′-end trimming after Cas6 cleavage of precursor crRNAs. Mature crRNAs in type III systems investigated to date typically have an 8 nt conserved repeat sequence tag at their 5′ end, and 3′-end trimming generally results in 2 or more distinct mature crRNAs derived from the same spacer sequence (19). In type III-B P. furiosus, for example, the 5′ end generated by Cas6 cleavage is maintained in mature crRNA, but crRNAs undergo further processing at the 3′ end to generate crRNAs of either ∼45 or ∼39 nt in length (41, 49). Similarly, Staphylococcus epidermidis (type III-A) has crRNAs that are 37, 43, and 72 nt in length (50). Walker et al. (28) reported that 3′ trimming of crRNAs in the type III-A S. epidermidis (Csm) complex is catalyzed by a trans-acting non-Cas nuclease, probably polyribonucleotide Phosphorylase. However, mature crRNAs detected here in the M. tuberculosis system were of uniform size (∼71 nt Figs. 1B and 2) and were comprised of an intact spacer sequence flanked by 8 nt 5′ and 28 nt 3′ repeat sequence tags (Fig. 2), the 3′ sequence tag forming a hairpin structure. M. tuberculosis crRNAs thus more closely resemble mature crRNA from type I systems, which are of uniform length and are comprised of intact spacer sequences flanked by 5′ and 3′ repeat sequence tags primary processing of precursor crRNA by Cas6 in M. tuberculosis appears to be sufficient to generate mature crRNA, with no further 3′-end trimming required. Mature crRNAs in E. coli (type I-E), for example, are ∼61 nt in length and have 8 nt 5′ and 20 nt 3′ repeat sequence tags (26, 54). The same mature crRNA structure is observed in type I-C systems, such as that in Bacillus halodurans and Mannheimia succiniciproducens (55, 56), and in type I-F systems, such as that in P. aeruginosa (22). The role of Cas6 in M. tuberculosis thus resembles that of the CasE (Cas6e) subunit in the E. coli type I-E system that is necessary and sufficient for pre-crRNA cleavage. The most likely explanation for the difference in mature crRNA structure between M. tuberculosis and other type III systems is that M. tuberculosis may lack the enzymes involved in 3′ end trimming present in other type III systems. The atypical structure of M. tuberculosis crRNAs may point to further differences in the mechanisms of invader silencing among type III systems and requires further investigation.

Cas6 is classified as a member of the Cas6-I-III super-family of endoribonucleases that contain a double RRM/ferredoxin fold. The endonuclease activity of M. tuberculosis Cas6 observed here was dependent on divalent metal ions Ca 2+ and Mn 2+ strongly promote Cas6 activity (Fig. 3B). Isothermal titration calorimetry assays further demonstrated that M. tuberculosis Cas6 binds Ca 2+ directly (Fig. 3C). Cas6 ion–dependent RNA cleavage has not been observed in other bacteria. For example, Cas6 in type III-B P. fiiriosus, and its Cas6 homologues Csy4 in type I-F Pseudomonas aeruginosa, and Cas6e in type I-E E. coli are reported to cleave CRISPR transcripts in a metal ion-independent manner, with addition of metal ions or EDTA having no effect on Cas6 cleavage activity in vitro (22, 25, 35). However, metal ion-dependent DNA cleavage has been observed in Cas5d, the B. halodurans Cas6 homologue (57). Metal ions are known to play a variety of roles in reaction mechanisms, including structural changes around the active site in some enzymes or acting as electrophilic activators for P-O and C-O bonds (58, 59). Further work is required to determine whether metal ions bind to M. tuberculosis Cas6 at its active site and how they affect Cas6 activity.

Residues involved in Cas6 cleavage are highly conserved among Cas6 homologues. M. tuberculosis Cas6, like T. thermophilus Cse3 and P. fiiriosus Cas6, is composed of duplicated ferrodoxin folds separated by a central cleft that contains a conserved glycine-rich loop (25, 60) (Fig. 3E). Previous reports have indicated that a highly conserved histidine residue located adjacent to the glycine-rich loop is an active-site residue critical for catalyzing cleavage of CRISPR repeat RNA (22, 35, 61). Here, mutation of the corresponding His99 and Gly295/Gly297 residues (located in different RRM/ferredoxin folds) to alanine abolished accurate Cas6 cleavage (Fig. 3F). Although the G295A/G297A mutant protein could not bind repeat RNA, the H99A mutant protein was still able to bind repeat RNA (Fig. 3G), confirming the importance of these 3 residues in Cas6 recognition and cleavage of its substrate RNA in M. tuberculosis.

Cas6 enzymes, a class of highly sequence- and structure-specific endoribonucleases involved in the maturation of crRNAs in type I and III CRISPR systems (34, 62), can broadly be divided into 2 classes based on their substrate specificity: 1) cleaving RNA substrates that have a hairpin structure and 2) cleaving RNA substrates that lack hairpin structures. If the hairpin structure is disrupted or if its terminal base sequence mutated, the cleavage activity of Cas6 enzymes that cleave RNA substrates with stem-loop structures is abolished. For example, disrupting the hairpin loop or mutating the bases of the hairpin stem abolishes the cleavage activity of S. epidermidis (type III-A) Cas6 (62). In species such as Pyrococcus horikoshii that cleave crRNAs that do not have a hairpin structure, accurate cleavage depends on the sequence of the crRNA itself, and specific base mutations will abolish cleavage (24, 63). The M. tuberculosis repeat sequence is predicted to contain 2 hairpins, located at its 5′ and 3′ ends. RNA footprinting indicated that the most important Cas6 cleavage site is at hairpin B, which is the larger of the 2 hairpins. Cas6 cleaves at the terminal base of hairpin B, generating a 28 nt RNA cleavage band. In contrast to the general situation previously described, although disrupting the structure of hairpin B or mutating its terminal bases resulted in the disappearance of the main 28 nt RNA cleavage product, the overall cleavage activity of Cas6 was not affected (Fig. 4B). However, mutating the sequence of the hairpin loop did not affect generation of the main 28 nt cleavage band. These results indicate that, although both the presence of a hairpin structure and its terminal base sequence are very important for accurate cleavage by M. tuberculosis Cas6, they are not required for Cas6 cleavage activity per se.

Extensive investigations of CRISPR/Cas prokaryotic immune systems in recent years have revealed significant levels of diversity between species in their protein components and transcriptional processes and have led to several reclassifications of CRISPR systems (6). Evidence presented here indicates that, although the M. tuberculosis CRISPR/Cas system is correctly classified as type III-A based on its major protein components, the structure of its mature crRNA more closely resembles that of type I systems in that the extensive 3′ end processing typical of type III systems is apparently not required. We postulate that M. tuberculosis may lack theenzymes responsible for 3′ end processing in other type III systems. As the enzymes involved in 3′ end processing mechanisms in other type III systems are identified, it may become appropriate to create a special subcategory within type III-A systems for M. tuberculosis.

In summary, we have systematically characterized mature crRNA structure and crRNA generation in M. tuberculosis, an ancient pathogen that remains a scourge on the global public health scene. Because M. tuberculosis is a slow-growing and experimentally intransigent pathogen, many aspects of its basic biology, and their impact on pathogenesis, are yet to be elucidated. The new understanding of the M. tuberculosis CRISPR/Cas system generated here will be of value in studies on the evolution and epidemiology of M. tuberculosis and lays a strong foundation for further investigation of the mechanisms of this atypical type III CRISRP/Cas system and evaluation of the potential of using elements of such a type III CRISPR/Cas system in genome editing.


References

A. J. Bogdanove, et al., Nucleic Acids Res. 46, 4845–4871 (2018), doi: 10.1093/nar/gky289.

A. C. Komor, A. H. Badran, D. R. Liu, Cell 168, 20–36 (2017), doi: 10.1016/j.cell.2016.10.044.

D. E. Paschon et al., Nat. Comm. 10, 1133 (2019), doi: 10.1038/s41467-019-08867-x.

X. S. Liu et al., Cell 172, 979–992 (2018), doi: 10.1016/j.cell.2018.01.012.

L. Villiger et al., Nat. Med. 24, 1519–1525 (2018), doi: 10.1038/s41591-018-0209-1.

A. Pickar-Oliver, C. A. Gersbach, Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019), doi: 10.1038/s41580-019-0131-5.

C. T. Charlesworth et al., Nat. Med. 25, 249–254 (2019), doi: 10.1038/s41591-018-0326-x.

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Porvair Sciences announce the Chromatrap Homogenizer Spin Column, which provides a cost-effective way to homogenize cells and tissues lysates in a single step. Developed as a quick, clean, and efficient alternative to syringe- and needle-homogenization techniques, the Homogenizer offers labs an easy-to-use, downstream sample-processing tool for a wide range of applications, including plasmid miniprep and midiprep and RNA extraction protocols. Good homogenization of cells and tissue lysates enables improvement in yield and quality of obtained plasmids and RNA for downstream applications. The unique proprietary bioshredding Vyon polymer contained within the column reduces lysate viscosity and captures insoluble debris by centrifugation, thereby eliminating the possibility of contamination.

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Chris Tachibana

Chris Tachibana is a science writer based in Seattle, USA, and Copenhagen, Denmark.


If a DNA of an organism is genetically modified, the resulting organism is called a "knockdown organism." 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". [1]

In a transient knockdown, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression through a variety of processes. Binding can occur either through the blocking of transcription (in the case of gene-binding), the degradation of the mRNA transcript (e.g. by small interfering RNA (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 or other RNase-H independent antisense). [1] [2]

The most direct use of transient knockdowns is for learning about a gene that has been sequenced, but has an unknown or incompletely known function. This experimental approach is known as reverse genetics. Researchers draw inferences from how the knockdown differs from individuals in which the gene of interest is operational. 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 through embryonic development. [3] The term gene knockdown first appeared in the literature in 1994 [4]

RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation. [5] Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (RISC). [6] 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 widely used as a laboratory technique for genetic functional analysis. [7] RNAi in organisms such as C. elegans and Drosophila melanogaster provides a quick and inexpensive means of investigating gene function. In C. elegans research, the availability of tools such as the Ahringer RNAi Library give laboratories a way of testing many genes in a variety of experimental backgrounds. Insights gained from experimental RNAi use may be useful in identifying potential therapeutic targets, drug development, or other applications. [8] RNA interference is a very useful research tool, allowing investigators to carry out large genetic screens in an effort to identify targets for further research related to a particular pathway, drug, or phenotype. [9] [10]

A different means of silencing exogenous DNA that has been discovered in prokaryotes is a mechanism involving loci called 'Clustered Regularly Interspaced Short Palindromic Repeats', or CRISPRs. [11] CRISPR-associated (cas) genes encode cellular machinery that cuts exogenous DNA into small fragments and inserts them into a CRISPR repeat locus. When this CRISPR region of DNA is expressed by the cell, the small RNAs produced from the exogenous DNA inserts serve as a template sequence that other Cas proteins use to silence this same exogenous sequence. The transcripts of the short exogenous sequences are used as a guide to silence these foreign DNA when they are present in the cell. This serves as a kind of acquired immunity, and this process is like a prokaryotic RNA interference mechanism. The CRISPR repeats are conserved amongst many species and have been demonstrated to be usable in human cells, [12] bacteria, [13] C. elegans, [14] zebrafish, [15] and other organisms for effective genome manipulation. The use of CRISPRs as a versatile research tool can be illustrated [16] by many studies making use of it to generate organisms with genome alterations.

Another technology made possible by prokaryotic genome manipulation is the use of transcription activator-like effector nucleases (TALENs) to target specific genes. [17] TALENs are nucleases that have two important functional components: a DNA binding domain and a DNA cleaving domain. The DNA binding domain is a sequence-specific transcription activator-like effector sequence while the DNA cleaving domain originates from a bacterial endonuclease and is non-specific. TALENs can be designed to cleave a sequence specified by the sequence of the transcription activator-like effector portion of the construct. Once designed, a TALEN is introduced into a cell as a plasmid or mRNA. The TALEN is expressed, localizes to its target sequence, and cleaves a specific site. After cleavage of the target DNA sequence by the TALEN, the cell uses non-homologous end joining as a DNA repair mechanism to correct the cleavage. The cell's attempt at repairing the cleaved sequence can render the encoded protein non-functional, as this repair mechanism introduces insertion or deletion errors at the repaired site.

So far, knockdown organisms with permanent alterations in their DNA have been engineered chiefly for research purposes. Also known simply as knockdowns, these organisms are most commonly used for reverse genetics, especially in species such as mice or rats for which transient knockdown technologies cannot easily be applied. [3] [18]

There are several companies that offer commercial services related to gene knockdown treatments.


Difference between micro RNA and short-interfering RNA and CRISPR Cas 9 system? - Biology

RNA-guided nucleases (RGNs) provide sequence-specific gene regulation through base-pairing interactions between a small RNA guide and target RNA or DNA. RGN systems, which include CRISPR-Cas9 and RNA interference (RNAi), hold tremendous promise as programmable tools for engineering and therapeutic purposes. However, pervasive targeting of sequences that closely resemble the intended target has remained a major challenge, limiting the reliability and interpretation of RGN activity and the range of possible applications. Efforts to reduce off-target activity and enhance RGN specificity have led to a collection of empirically derived rules, which often paradoxically include decreased binding affinity of the RNA-guided nuclease to its target. We consider the kinetics of these reactions and show that basic kinetic properties can explain the specificities observed in the literature and the changes in these specificities in engineered systems. The kinetic models described provide a foundation for understanding RGN targeting and a necessary conceptual framework for their rational engineering.

Present address: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA


CONCLUSION

The startling discovery that nucleotide sequence information can cross cell boundaries in the form of regulatory RNA necessitates a revision of our understanding of animals. The movement of mobile RNA between cells is minimally a new form of cell-to-cell communication within animals. The ability of foreign mobile RNA to enter and affect gene regulation in some animals hints at intimate communication between an animal and its environment. Extending this concept to an extreme, we can imagine a scenario where an animal cell responds to imported RNA that was exported from a cell in another organism. Much work remains to be done to discover how natural selection has favored organisms that can transport RNA across membranes to evolve gene regulatory interactions across the animal kingdom.