Posted by on June 20, 2020 10:32 am
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collector’s Edition review|RNA-targeting CRISPR systems-from the metagenome to the RNA regulation

author | Magnolia of 枻

responsible for encoding | XI

< p>to the intracellular RNA as a target of Biological Research in DNA-targeting research flourished after the start, and in the early 21st century breakthrough.

this is one of two major breakthroughs, one is the MS2 bacteriophage capsid protein(VCP), which binds a specific RNA stem-loop structure is widely used for intracellular mRNA imaging and stability of regulation; the other is RNA interference(RNAi)technology, which is efficient and specific inhibition of gene expression the ability to make it into RNA functional studies of the weapon.

subsequent ten years, the VCP and RNAi lead the trend, and new technologies are constantly emerging for RNA targeting research to bring unlimited vigor. Among these, RNA-targeting CRISPR systems RCas due to its huge potential to attract people attention.

its origin is concerned, the CRISPR system is a prokaryotic resistance to exogenous virus and phage invasion of the Acquired Immune System.

the basic principle is the Cas protein and the guide RNA(crRNA forming a complex, and then binding and destruction with a guide RNA complementary to the target sequence. Depending on the system, the target sequence may be double-stranded DNA, dsDNA, or may be single-stranded DNA(ssDNA or single-stranded RNA(ssRNA)。

overall, the CRISPR system can be divided into two categories, Class 1 system functional complexes require multiple Cas proteins participate; a Class 2 system only needs a single Cas protein involved. Taking into account the system’s simplicity, the researchers more attention to the Class 2 system transformation, and in the study of Early used for DNA targeting research and genome editing and has achieved fruitful results.

in the target DNA to the research has made many advances, after taking into account the CRISPR system is also targeted to the RNA of ability, researchers began to pay attention to the RCas system of the excavations and reconstruction, so as to promote target RNA to study the rapid development.

not long ago, from the University of California, San Diego at UCSD cell and Molecular Medicine, Department ofGene W. YeoDr.Nature Cell BiologyentitledRNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineeringreview.

article of the RCas system of the research progress has combed from the RCas system of the filtering, transformation and application of various aspects are summarized, which we have systematic awareness of the RCas system has an important addition, the researchers also pointed out that the areas facing difficulties and challenges, so as future research provides ideas and insights.

the author of the first of the RCas system of screening, verification, and species were summarized(Figure 1a)。 Currently the most widely used of the RCas system can be divided into two categories, one is the DNA-targeting CRISPR system transformation, enabling the RNA targeting and regulation, but such transformation of the system there are still unsatisfactory place.

another category is the specific recognition of ssRNA CRISPR system, such RCas system of discovery is mainly dependent on bio-Informatics-based metagenomic mining and screening.

through continuous debugging, the researchers build a more comprehensive bioinformatics filtering process, resulting in the metagenome found in the many potential of the RCas system.

after passing through the maturity of the test process, the researchers of the candidate system in the Cas effectShould the activity of the protein, the structure of the domain, the crRNA features and target sequence characteristics of System Analysis, eventually found a wide application value of the RCas system.

for now, the RCas system to a 2 class system of the type II Cas9 and Type IV Cas13 of the most widely used(Figure 1b)。 Traditional CRISPR-Cas9 system is widely used for DNA targeting as the basis of genome editing research.

then through the Supplement contain the PAM sequence of the oligonucleotide chain, such the CRISPR-Cas9 system can be implemented in RNA targeting, the final recognition and cleavage of target ssRNA. span>

with Cas9 to a different, so far found Cas13 system only the specific recognition and targeting of ssRNA, and Cas13-crRNA complex in the recognition of the target sequence, it will be no difference in the cutting and degradation of non-target ssRNA, this feature was later widely used for Molecular Diagnostic study.

in addition to the RCas outside of the system, the researchers for other types of RNA-targeting techniques can also be a simple summary of(Figure 1c), including prokaryotes Ago system, antisense oligonucleotide technologies, ASOs, as well as the recent development of CIRTS technology, such technologies with the RCas system compared each have advantages and disadvantages, equally worth our attention.

Figure 1 RCas system of screening and verification strategy a, the RCas system of the species b and its target RNA to the system c

to the RCas system of the screening process and species were summarized after the, author from basic research and industry, two aspects of the RCas applications are summarized.

on the basis of research, the RCas system can be widely applied to RNA detection and labeling, RNA expression and regulation, epigenetic modification, RNA editing and localization and RNA programming and other areas of2)

in fact, in RNA research, there is also a variety of other techniques, such technique in multiple research fields has its uniqueness. Such as ASOs in the RNA expression of the regulation on the equally well; and in the RNA editing in the field, does not depend on the RCas of the new editing technology is also very competitive.

of course, the RCas system also has its uniqueness, such as Cas9-METTL13/METTL14/ALKBH5/FTO fusion proteins can be achieved on the RNA m6A methylation in the regulation, the RCas system can also be used for RNA-binding protein function research and synthetic biology of RNA programming.

Figure 2 RCas system on the basis of Biological Research in the application

in addition to the basic research in addition, the RCas system in the industry the same potential, wherein the nucleic acid detection-based disease diagnosis is particularly striking.

Cas13-crRNA complex in the recognition of the target sequence after the other ssRNA were no difference in cutting ability is researchers widely used include the walled card, dengue fever and other diseases diagnosis; Cas13 this characteristic can also be modified for antiviral R & D, which the viral infection has important significance.

the researchers also found that the transformation of the RCas system may also promote the cytotoxic RNA to degradation of the polymer, which includes neurodegenerative diseases, including the treatment of various diseases has provided new ideas.

finally, the researchers also of the RCas system of the application process need to pay attention to the key points are summarized: at the molecular level this includes how to choose according to their needs different RCas system, whether you need to add the extra effect of the factor to construct a fusion protein, the fusion protein construct of the policy, the crRNA to the target sequence of choice, the RCas system of the delivery strategy, and so on; at the cellular level you need to consider the RCas system of active regulation, the security of the system and different component ratio and the like���

in addition, taking into account the RCas system in the disease treatment process requires continuous expression, which is the biggest challenge is immunogenic may trigger the immune response and potential toxicity.< span class="bjh-br">

overall, although there are a variety of shortcomings, but considering the RCas research is still in its infancy, current research progress remains impressive. We have reason to believe that the future for RCas based tool development and application to explore will achieve more results, which will further enrich the target RNA to the library of tools and expand its application scope.

in addition, related research will also allow us to RNA-targeting studies have a more comprehensive understanding and awareness.

prepress person: ke

Published at Sat, 20 Jun 2020 14:31:47 +0000