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| | __NOTOC__ | | __NOTOC__ |
| − | <partinfo>BBa_K1431401 short</partinfo> | + | <partinfo>BBa_K1431402 short</partinfo> |
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| − | <partinfo>BBa_K1431401 SequenceAndFeatures</partinfo> | + | <partinfo>BBa_K1431402 SequenceAndFeatures</partinfo> |
| − | | + | |
| − | | + | |
| − | === Introduction ===
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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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| − | <blockquote>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.<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>
| + | |
| − | 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'''
| + | |
| − | </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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| − | 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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| − | | + | |
| − | 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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| − | 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 === | | === Designing the Sequence === |
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| | The whole process can be divided into the following steps: | | The whole process can be divided into the following steps: |
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| | ==== Conserved Sequence Analysis ==== | | ==== Conserved Sequence Analysis ==== |
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| | ==== Strip out sequences without PAM ==== | | ==== Strip out sequences without PAM ==== |
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| − | {| class="wikitable"
| |
| − | ! colspan="11" | Supplementary Table 1 - Base Percentage of HIV-1 Aligned Genome 730bp-752bp
| |
| − | |-
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| − | |
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| − | | A %
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| − | | G %
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| − | | C %
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| − | | T %
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| − | | Empty %
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| − | | Non Empty %
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| − | | A(Corrected)
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| − | | G(Corrected)
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| − | | C(Corrected)
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| − | | T(Corrected)
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| − | |-
| |
| − | | 730
| |
| − | | 0
| |
| − | | 0
| |
| − | | 0
| |
| − | | 56.47
| |
| − | | 43.53
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| − | | 56.47
| |
| − | | 0.00%
| |
| − | | 0.00%
| |
| − | | 0.00%
| |
| − | | 100.00%
| |
| − | |-
| |
| − | | 731
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| − | | 0
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| − | | 55.88
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| − | | 0
| |
| − | | 0.59
| |
| − | | 43.53
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| − | | 56.47
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| − | | 0.00%
| |
| − | | 98.96%
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| − | | 0.00%
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| − | | 1.04%
| |
| − | |-
| |
| − | | 732
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| − | | 0
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| − | | 0
| |
| − | | 0
| |
| − | | 56.47
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| − | | 43.53
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| − | | 56.47
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| − | | 0.00%
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| − | | 0.00%
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| − | | 0.00%
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| − | | 100.00%
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| − | |-
| |
| − | | 733
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| − | | 0
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| − | | 54.71
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| − | | 0
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| − | | 1.18
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| − | | 43.53
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| − | | 55.89
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| − | | 0.00%
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| − | | 97.89%
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| − | | 0.00%
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| − | | 2.11%
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| − | |-
| |
| − | | 734
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| − | | 0
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| − | | 0
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| − | | 0
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| − | | 58.24
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| − | | 41.76
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| − | | 58.24
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| − | | 0.00%
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| − | | 0.00%
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| − | | 0.00%
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| − | | 100.00%
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| − | |-
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| − | | 735
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| − | | 56.47
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| − | | 0.59
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| − | | 0.59
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| − | | 0.59
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| − | | 41.76
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| − | | 58.24
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| − | | 96.96%
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| − | | 1.01%
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| − | | 1.01%
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| − | | 1.01%
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| − | |-
| |
| − | | 736
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| − | | 0
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| − | | 1.18
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| − | | 57.06
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| − | | 0
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| − | | 41.76
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| − | | 58.24
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| − | | 0.00%
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| − | | 2.03%
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| − | | 97.97%
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| − | | 0.00%
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| − | |-
| |
| − | | 737
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| − | | 1.18
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| − | | 57.06
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| − | | 0
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| − | | 0.59
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| − | | 41.18
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| − | | 58.83
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| − | | 2.01%
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| − | | 96.99%
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| − | | 0.00%
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| − | | 1.00%
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| − | |-
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| − | | 738
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| − | | 60
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| − | | 0
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| − | | 0
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| − | | 0
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| − | | 40
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| − | | 60
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| − | | 100.00%
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| − | | 0.00%
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| − | | 0.00%
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| − | | 0.00%
| |
| − | |-
| |
| − | | 739
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| − | | 0.59
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| − | | 0
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| − | | 58.82
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| − | | 0
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| − | | 40
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| − | | 59.41
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| − | | 0.99%
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| − | | 0.00%
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| − | | 99.01%
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| − | | 0.00%
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| − | |-
| |
| − | | 740
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| − | | 0
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| − | | 0
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| − | | 0
| |
| − | | 0
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| − | | 100
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| − | | 0
| |
| − | |
| |
| − | |
| |
| − | |
| |
| − | |
| |
| − | |-
| |
| − | | 741
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| − | | 0
| |
| − | | 0
| |
| − | | 0
| |
| − | | 0
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| − | | 100
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| − | | 0
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| − | |
| |
| − | |
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| − | |
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| − | |
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| − | |-
| |
| − | | 742
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| − | | 0.59
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| − | | 0
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| − | | 1.18
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| − | | 58.24
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| − | | 40
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| − | | 60.01
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| − | | 0.98%
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| − | | 0.00%
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| − | | 1.97%
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| − | | 97.05%
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| − | |-
| |
| − | | 743
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| − | | 0
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| − | | 0
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| − | | 60
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| − | | 0
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| − | | 40
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| − | | 60
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| − | | 0.00%
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| − | | 0.00%
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| − | | 100.00%
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| − | | 0.00%
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| − | |-
| |
| − | | 744
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| − | | 0
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| − | | 1.18
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| − | | 58.82
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| − | | 0
| |
| − | | 40
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| − | | 60
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| − | | 0.00%
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| − | | 1.97%
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| − | | 98.03%
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| − | | 0.00%
| |
| − | |-
| |
| − | | 745
| |
| − | | 0
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| − | | 58.82
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| − | | 1.18
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| − | | 0
| |
| − | | 40
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| − | | 60
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| − | | 0.00%
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| − | | 98.03%
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| − | | 1.97%
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| − | | 0.00%
| |
| − | |-
| |
| − | | 746
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| − | | 0.59
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| − | | 0
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| − | | 59.41
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| − | | 0
| |
| − | | 40
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| − | | 60
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| − | | 0.98%
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| − | | 0.00%
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| − | | 99.02%
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| − | | 0.00%
| |
| − | |-
| |
| − | | 747
| |
| − | | 0.59
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| − | | 59.41
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| − | | 0
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| − | | 0
| |
| − | | 40
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| − | | 60
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| − | | 0.98%
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| − | | 99.02%
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| − | | 0.00%
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| − | | 0.00%
| |
| − | |-
| |
| − | | 748
| |
| − | | 0.59
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| − | | 59.41
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| − | | 0
| |
| − | | 0
| |
| − | | 40
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| − | | 60
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| − | | 0.98%
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| − | | 99.02%
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| − | | 0.00%
| |
| − | | 0.00%
| |
| − | |-
| |
| − | | 749
| |
| − | | 0
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| − | | 58.82
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| − | | 0.59
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| − | | 0.59
| |
| − | | 40
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| − | | 60
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| − | | 0.00%
| |
| − | | 98.03%
| |
| − | | 0.98%
| |
| − | | 0.98%
| |
| − | |-
| |
| − | | 750
| |
| − | | 0.59
| |
| − | | 0.59
| |
| − | | 58.24
| |
| − | | 0.59
| |
| − | | 40
| |
| − | | 60.01
| |
| − | | 0.98%
| |
| − | | 0.98%
| |
| − | | 97.05%
| |
| − | | 0.98%
| |
| − | |-
| |
| − | | 751
| |
| − | | 60
| |
| − | | 0
| |
| − | | 0
| |
| − | | 0
| |
| − | | 40
| |
| − | | 60
| |
| − | | 100.00%
| |
| − | | 0.00%
| |
| − | | 0.00%
| |
| − | | 0.00%
| |
| − | |-
| |
| − | | 752
| |
| − | | 59.41
| |
| − | | 0.59
| |
| − | | 0
| |
| − | | 0
| |
| − | | 40
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| − | | 60
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| − | | 99.02%
| |
| − | | 0.98%
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| − | | 0.00%
| |
| − | | 0.00%
| |
| − | |}
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| | | | |
| − | ==== Select gRNA sequences with the best theoretical quality ==== | + | ====Select gRNA sequences with the best theoretical quality==== |
| | + | |
| | + | |
| | + | |
| | + | |
| | | | |
| − | {| class="wikitable"
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| − | ! colspan="5" | HIV-1 Quasi-Conservative gRNAs(Useful)
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| − | |-
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| − | | Sequence
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| − | | Rating(Zhang)
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| − | | Rank(Church)
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| − | | Free Energy(Approx.)
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| − | |
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| − | |-
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| − | | GTGTGGAAAATCTCTAGCAGTGG
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| − | | 71
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| − | | -
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| − | | -1.4
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| − | | rowspan="2" | HIV1_REF_2010
| |
| − | |-
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| − | | TCTAGCAGTGGCGCCCGAACAGG
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| − | | 97
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| − | | -
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| − | | -1.3
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| − | |}
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| | | | |
| | ===Source=== | | ===Source=== |
| | | | |
| − | Conserved Region of the HIV-1 Genome from the NIH HIV-1 Sequence Database
| + | AB010289-AB078031 in HBV_aligned Database |
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| | ===References=== | | ===References=== |
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.
