Team:Austin LASA/Design

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h(g.Page, {title: 'Design, Development, and Results', prev: 'https://2018.igem.org/Team:Austin_LASA/Description', next: 'https://2018.igem.org/Team:Austin_LASA/Notebook', selector: [2, 1]},
  h(g.Section, {title: 'What will be used to detect HIV?'},
    p('Because our final Cas12a detection assay relies on sequence recognition between the crRNA spacer sequence and a target strand, we only needed to choose specific sequences from HIV’s genome to work with.'),
  ),
  h(g.Section, {title: 'Choosing a Gene'},
    p('In the end, we chose to work with one of HIV’s genes to simplify our work in the lab. Because HIV is a BSL3 organism, we were unable to work with HIV or any HIV-infected human cells in the lab per iGEM’s Biosafety Standards. Instead, we worked with synthetic viral DNA that we ordered as gblocks through IDT.'),
    p('We ultimately focused on Rev, a non-hazardous gene in HIV’s genome which codes for a transportation protein in HIV. The sequence we found for Rev was specifically from HIV1, and as a result, our subsequent work in the lab focused on the detection of HIV-1.'),
    p('Rev served as the viral sequence we would attempt to amplify with loop-mediated isothermal amplification (LAMP) and subsequently detect with our Cas12a assay.')
  ),
  h(g.Section, {title: 'Designing crRNA'},
    p('Once we decided on an HIV gene to detect, we went ahead and designed crRNA sequences for our Cas12a assay. crRNA sequences are the basis of our detection assay. However, Cas12a crRNA design is still a field of much study, and it was very difficult to find specific information on how to design crRNA. Ultimately, the design of our crRNA was broken up into two components: the hairpin sequence and spacer sequence.'),
    p('We decided on two hairpin sequences, one for AsCas12a and one for LbCas12a. Our LbCas12a sequence was taken from the Doudna Lab. Our AsCas12a sequence was taken from the Finkelstein Lab. We would test our AsCas12a crRNA with purified AsCas12a enzyme, and we would later attempt to clone a gene circuit expressing LbCas12a to use with our LbCas12a crRNA in our cellular reagents.'),
    p('We used several criteria when choosing our spacer sequence. The most important criteria for choosing our spacer sequence was that it needed to be around 24bp following Cas12a’s PAM site, TTTN. From there, we distinguished between spacer sequences based on the following:'),
    h('ul', null,
      h('li', null, 'GC content'),
      h('li', null, 'Dimer formation'),
      h('li', null, 'Similarities to Cas9 sgRNA design')
    ),
    p('Ultimately, we designed 7 different crRNA sequences, and ultimately used 4.'),
  ),

);