Questions:

1. Which others promoters will work on this plasmid?
2. How would your promoter design change if we were going to express the gene products
   in vitro instead?
   About Target Finder Results:
3. How many hits does the target finder find if you use the 5’GG requirement?
4. Which hit is closest to the start codon?
5. Why is this hit NOT chosen as the top scoring hit?
6. What makes the top scoring hit so successful?
   CRISPR In General
7. Generally speaking, what makes a good CRISPR guide RNA?
8. What is the difference between Homology Directed Repair and Non-Homologous End
   Joining?
   CRISPR Plasmid Design
   TASKS:
9. Design a single guide RNA (sgRNA) that will generate an INDEL in exon 1 of Gr21a,
   1
   and disable the expression of the gene. IN DROSOPHILA ZYGOTES
10. Build a plasmid containing
    a. sgRNA built in task 1
    b. Cas9, and a promoter that makes sense for our protocol.

We need a guideRNA (sgRNA) and Cas9 (protein) expressed in drosophila zygotes.
Easiest way is to put them both on a plasmid , and shuttle the plasmid into Drosophila zygote.
2
The CRISPR-Cas9 complex gets assembled after transcribed (and translated for Cas9) from
the plasmid.
The CRISPR-Cas9 complex will induce a double strand break (Ideally in both chromosomes),
which will be repaired by Non-Homologous End Joining (NHEJ)
Homologous Recombination (HR) is another way the cell can repair a double strand break. How the cell decides what
decision to make here depends on the severity of the damage at the DNA strand break site
Plasmid Into Cell
Also, remember we need to include a promoter sequence on
the plasmid, able to drive expression of Cas9 and sgRNA.
Using this blog, I found a promoter that should work. It is
important that the promoter works in drosophila, since this is
our target organism. I’m choosing Ac5.
Plasmid can be transfected using Lipofectamine
1
INDEL – a small (1~5nt) Insertion/Deletion that can be sufficiently repaired by NHEJ
2 Plasmid -small circular DNA strand containing nucleotide sequence you want to put in a cell
Mutagenesis via indel formation
Tip 1:
Tip: To disrupt Gr21a, we want to generate a frameshift in an
early exon.
Remember, the goal here is to disturb the open reading frame
that encodes the functional Gr21a protein. Look for a region
ideally in Exon 1, the earlier the better.
Decorated Fasta of Nucleotide sequence of Gr21a
http://flybase.org/decoratedfasta/FBgn0041250
Tip 2:
Intergenic regions make bad target sites!

• Because a lot of intergenic regions are not under
  selection pressure, these regions are likely to vary slightly
  in sequence between different flies. This variability has
  the potential to make a gRNA targeted to an intergenic
  region fail
  Tip 3:
  Look for all the PAM sequences in the region you want to target
  (Exon 1)
  https://www.youtube.com/watch?v=iSEEw4Vs_B4&t=3s
  Tip 4:
  This tool can find a good target sequence in exon 1 for you
  Targetfinder.flycrispr.neuro.brown.edu
  2 Metrics for scoring a CRISPR guide RNA
  Efficiency (E) score:
  “Does this target effectively target the gene of interest?” Biases towards targets early in
  sequence
  Specificity (S) Score
  “Do other binding sites (Off Targets) interfere with this site?”
  A high ranking generally Maximizes efficiency and minimizes off target sites (Max Specificity)
  (CRISPR-Era is a similar algorithm to what we will actually use today. Similar enough to make the point of “Efficiency of target
  binding” vs “Off Target Effect avoidance”. The math is a little different, but the overarching idea is the same.)
  In TargetFinder, paste Exon 1’s nucleotide sequence
  Use the most stringent query for best target sequence
  “CRISPR targets with 5′ GG “
• How many hits does the target finder find if you use the 5’GG requirement?

– Which hit is closest to the start codon?

Use the “Design Experiment feature to get the sequence for the top scoring hit. This is
what you should see:
In benchling, you can manipulate nucleotide sequences to build plasmids. This
sequence then gets sent to a lab where it is actually made.
Paste the sequence for the ACS promoter, the Cas9 protein, and the Sense oligo
into benchling.
Example of semi-final Plasmid. (actual plasmid will include a few other regulatory
elements, but these are outside of the scope of this discussion)
https://benchling.com/s/seq-Gj3XcMT3YuD5YG1Fqqyk
Protocol built from this:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3714591/