Difference between revisions of "Part:BBa K1431401"

Revision as of 18:47, 17 October 2014

One gRNA Sequence for HIV-1

This is a gRNA for the HIV-1 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 HIV 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.

Characterization

Usage and Biology

Sequence and Features