Part:BBa_K1431403
gRNA2 for HBV
This is a gRNA2 for the HBV Virus, designed by the 2014 iGEM Team SUSTC-Shenzhen.
Here, we engineer the type II CRISPR system to come true double strands break with gRNA in hela cells.We show that this process relies on CRISPR two specificity components:gRNA and its binding target-sequence.
In this part, we put on a high-specificity gRNA of HBV with more detail in design.
In addition ,this part use efficient BbsI restriction site for easily used.
Introduction
CRISPR(Clustered Regularly Interspaced Short Palindromic Repeat)/Cas System is a hot topic for biology research these days. Recently we see dozens of papers published in top journals addressing this intersting field. In case you are not familiar with it, I quoted those lines full of jargons from Wikipedia:
CRISPRs are DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a virus.
The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic elements like RNAi in eukaryotic organisms.
Since 2012, the CRISPR/Cas system has been used for gene editing (silencing, enhancing or changing specific genes) that even works in eukaryotes like mice and primates. By inserting a plasmid containing cas genes and specifically designed CRISPRs, an organism's genome can be cut at any desired location.
-Wikipedia
In short, CRISPR/Cas System is a tool to edit genes in live cells. Similar tools include TALEN(Transcription activator-like effector nuclease) and ZFN(Zinc Finger Nuclease). But CRISPR/Cas is superior than those methods in that CRISPR/Cas is guided by short RNA chain (~23bp), which is obviously easier to synthesize.
Moreover, TALENs require a significantly longer time to construct[http://indepth.systembio.com/cas9-crispr-faq/what-is-the-difference-between-cas9-crispr-and-talen SystemBio].
CRISPR gRNA Basics
As mentioned above, CRISPR/Cas9 Systems need a gRNA(Guide RNA) sequence to identify the target[http://www.nature.com/nprot/journal/v8/n11/full/nprot.2013.143.html ZhangFCas]. The gRNA is a 23bp long RNA beginning with a 3bp PAM(Protospacer Adjacent Motif) sequence. To effectively and specifically target a gene, the remaining 20bp of gRNA have to match the target sequence strictly. According to [http://crispr.mit.edu/about ZhangTool], the approximate quality of gRNA can be denoted as
.
Remember here that this equation bases only on an approximation of experimental data, and may differ from the actual situation.
Advantage
Why do we design plasmids that only carry gRNA sequence without Cas9?
One main thought of our project is to achieve the transferring of Cas9 system and gRNA sequences into human body separately at different period of time. As you know, there are two parts in our CRISPER system, one is the Cas9 proteins, and the other is the gRNA. To begin with, Cas9 system will be stably transfected into human cells firstly, remaining inactivated and just waiting for orders. While the transportation of gRNA will not be performed until the diseases have developed, and types of retrovirus that cause these diseases are confirmed. Then, since the type of the pathogenic retrovirus has been known at this moment, we can design a gRNA that can specifically recognize the very type of retrovirus, and transfer it into human body. Finally these gRNA will combine with activated Cas9 proteins, guiding them to the genome sequence of retrovirus, at last destroying it. By this way, we can achieve a more specific introduction of gRNA into human cells, specifically targeting for the very type of retrovirus that caused the disease. The separated transportations of the two parts may also partially overcome the difficulties of radical cure, always met in usual treatment, due to the high variability of retrovirus.
Characterization
Usage and Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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