Part:BBa_K1431401:Design

One gRNA Sequence for HIV-1

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Design Notes

Specificity and efficiency of the gRNA
2nd structure of the gRNA
Endonuclease Sites

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 <math>\prod\_{e\in{\mathcal{M}}}\left(1-\space W[e]\right)\times\frac{1}{\left(\frac{(19\space-\space\bar{d})}{19}\times4\space+\space1\right)}\times\frac{1}{n^2\_{mm}}</math>

Designing the Sequence

We used a method derived from the method described in the paper by Feng Zhang^{[http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2647.html ZhangFgRNA]}.

Source