Highlighted Reports on the CRISPR Cas System!


Selected papers for CRISPR Cas!

Segal and Meckler in 2013 state in ‘Genome Engineering at the Dawn of the Golden Age,’ that, “In summary, compared with ZFNs, TALENs can be designed for more targets, are more likely to be active on their targets, display higher on-target activity, and display lower off-target activity. New assembly methods and the current freedom from patent limitations have made TALENs more accessible and able to be produced in greater quantities than has been possible with ZFNs.” However, the CRISPR Cas system has the potential to surpass this.

The impact for the future of CRISPR Cas is reflected by the following statement using the words of Seagal and Meckler: CRISPR Cas: The Nuclease of the Future?  Even as TALENs were displacing ZFNs as the tool of choice for generating site-specific double strand breaks in DNA, a newly characterized natural endonuclease system threatened to make both obsolete. “ Jinek et al. in 2012 reported that the CRISPR Cas system from bacteria and archaea mediates DNA cleavage by using simple base pairing to specify the cut site. The CRISPR Cas systems provide invader-specific, adaptive and heritable immunity against viruses and plasmids by protecting against invading nucleic acids by guiding the molecular scissors to cleave non-host sequences that have been seen before into smaller pieces. The CRISPR Cas system is a novel microbial defense system found in half of the bacterial and almost all archaeal genomes sequenced. To function, the system needs the CRISPRs and the Cas proteins and relies on non-translated RNAs to track and inactivate invasive genetic elements to protect the cells genomic integrity. Jinek et al. discovered that one version of the CRISPR Cas system can be simplified to a single protein, Cas9, along with a single chimeric guide RNA to enable effective cleavage.

There are three types of CRISPR Cas systems, type I, II and III. Type I and II share specialized Cas endo-nucleases that process the pre-crRNAs, and once mature, each crRNA assembles into a large multi-Cas protein complex that recognizes and cleaves nucleic acids complementary to the crRNA. However, type II processes pre-crRNAs using a different mechanism in which a trans-activating crRNA (tracrRNA) complementary to the repeat sequences in pre-crRNAs triggers processing of the double-stranded RNA-specific ribonuclease RNAse II in the presence of the Cas9 protein. The Cas9 protein appears to be the responsible part for crRNA-guided silencing of foreign DNA. The type II CRISPR system works by incorporating short exogenous DNA sequences from the invading pathogen into specif loci of the host genome. At the time of transcription, these sequences are processed into crRNAs via pre-crRNAs. The crRNAs act as a guide for the Cas9 nuclease machinery. The Cas9 nuclease cleaves and inactivates the foreign DNA. The system requires two more pieces to function. A tracrRNA that forms base-pairs with the crRNA provides the substrate for the host’s ribonuclease RNase III. This system can identify DNA sequences that are complementary to the crRNA and degrade them. 

Cong et al. in 2013 showed that this three-component system made up of the

(1)  guide RNA (crRNA) that hybridizes to the target DNA,

(2)  the protein nuclease Cas9 that cleaves the target DNA (bacterial Cas9), and

(3) a linker RNA that brings the nuclease to the guide RNA (the tracrRNA) is sufficient to mediate efficient genome editing in human cells.

On the other hand, Mali et al. showed that a two-component system that included the

(1)  Cas9 protein and

(2)  a guide RNA consisting of a crRNA-tracrRNA hybrid molecule was also sufficient.

By designing guide RNAs for five DNA target sites Jinek et al. were able to demonstrate that the system had the ability to cleave all the sites in vitro using only purified Cas9 and the guide RNAs. The target site needed to contain a NGG motif immediately adjacent to the complementary region, providing a CRISPR Cas nuclease site at approximately every 8 bp in random DNA.

Instead of engineering new proteins for each cleavage site, researchers now need only to synthesize a new guide RNA to program the nuclease.

A comparison of the TALEN with the CRISPR Cas system reveals that a typical TALEN requires two new ~1,800-bp repeat coding regions to be assembled for each new target site, in contrast, a CRISPR Cas system would require just one new 20-nucleotide (nt) DNA-complementing region of the ~100-nt guide RNA, allowing any investigator to create hundreds or thousands of nucleases at low cost.

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Reference

Cong, L, Ran, FA, Cox, D, Lin, S, Barretto, L, Habib, N et al. (2013). Multiplex genome engineering using CRISPR/Cas Systems. Science, e-pub ahead of print 3 January 2013.

Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337:816–821. [PubMed]

Mali, P, Yang, L, Esvelt, KM, Aach, J, Guell, M, Di-Carlo, JE et al. (2013). RNA-guided human genome engineering via Cas9. Science, e-pub ahead of print. 3 January 2013.

David J. Segal and Joshua F. Meckler; Genome Engineering at the Dawn of the Golden Age Annu. Rev. Genomics Hum. Genet. 2013. 14:135–58. First published online as a Review in Advance on May 20, 2013. The Annual Review of Genomics and Human Genetics is online at genom.annualreviews.org.

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Categories: Bioanalysis, BNA RNA, BNAs, Cas9, Cellular Reprogramming, CRISPR Cas, Deep sequencing, DNA, DNA Analysis, DNA Editing, DNA finger printing, DNA Structure, DNA Synthesis, Epigenetics, Gene Editing, Gene Expression, Gene families, Genetics, Genome, In Vitro Transcription, IVT, LincRNA, lncRNA, Long noncoding RNA, non-coding RNAs, Nucleoprotein, Proteins, Regulatory RNA, RNA, RNA Editing, RNA FISH, RNA Structure, RNA Synthesis, RNA World

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