Difference between revisions of "Part:BBa K1431403:Design"
 
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<partinfo>BBa_K1431403 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1431403 SequenceAndFeatures</partinfo>
  
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=== Designing the Sequence ===
  
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=== Introduction ===
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We used a method derived from the method described in the paper by Feng Zhang<sup>[http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2647.html ZhangFgRNA]</sup>.
  
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'''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:
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The whole process can be divided into the following steps:
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<blockquote>CRISPRs are DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of &quot;spacer DNA&quot; from previous exposures to a virus.<br>
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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.<br>
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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.<br>
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'''-Wikipedia'''
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</blockquote>
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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.<br>
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==== Conserved Sequence Analysis ====
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Moreover, TALENs require a significantly longer time to construct<sup>[http://indepth.systembio.com/cas9-crispr-faq/what-is-the-difference-between-cas9-crispr-and-talen SystemBio]</sup>.
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=== CRISPR gRNA Basics ===
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We first extracted all conserved regions from the NIH HIV-1 Reference Genome. In this step, we found around 10 alternatives for the next process. Here all screening processes are done in a per-strain basis because of the high mutability of the HIV-1 virus.
  
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As mentioned above, CRISPR/Cas9 Systems need a gRNA(Guide RNA) sequence to identify the target<sup>[http://www.nature.com/nprot/journal/v8/n11/full/nprot.2013.143.html ZhangFCas]</sup>. 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 
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==== Strip out sequences without PAM ====
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https://static.igem.org/mediawiki/parts/2/23/Equation-crispr.png .
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Remember here that this equation bases only on an approximation of experimental data, and may differ from the actual situation.
 
  
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=== Designing the Sequence ===
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====Select gRNA sequences with the best theoretical quality====
  
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We used a method derived from the method described in the paper by Feng Zhang<sup>[http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2647.html ZhangFgRNA]</sup>.
 
  
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The whole process can be divided into the following steps:
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===Source===
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AB010289-AB078031 in HBV_aligned Database
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===References===

Latest revision as of 03:47, 18 October 2014

gRNA2 for HBV


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]

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].

The whole process can be divided into the following steps:

Conserved Sequence Analysis

We first extracted all conserved regions from the NIH HIV-1 Reference Genome. In this step, we found around 10 alternatives for the next process. Here all screening processes are done in a per-strain basis because of the high mutability of the HIV-1 virus.

Strip out sequences without PAM

Select gRNA sequences with the best theoretical quality

Source

AB010289-AB078031 in HBV_aligned Database

References