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:
1. 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
2. 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)
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/
Arrangement, with regards to composing, requires the creator to ace the expertise of remaining on theme for the sum of the paper (Jones, 2016). Staying on subject and just including significant data to the paper can be troublesome however will make a durable and efficient paper. The association and arrangement of the paper ought to be evident in the theme, issue, reason and the outlines the essayist incorporates into the insightful composition; keeping coherence of subject all through those areas of the paper will demonstrate legitimate arrangement. One approach to guarantee coherence is to make a proposal and fitting examination inquiries to sealing said theory. The creator will keep on alluding back to the proposition and questions when investigating the sources, writing, and information being incorporated into the paper. Accomplishing arrangement will help convince perusers and increment the likeliness of perusing perception by exhibiting sharp spotlight on tending to the postulation and addressing the examination questions explicitly.
Article One
Shahwan created an investigation finished in 2016 with respect to and fittingly titled, “Connection between ‘Power-Distance’ and Employee Performance in Multi-National Organizations” which planned for giving bosses some understanding into precisely surveying representative execution in a multi-social business.
Point
This present investigation’s point was to set up the impacts of “intensity separation” and the effect it has on representative execution. Power separation is term authored by Geer Hofstede when he set up a structure from which to dissect societies (Shahwan, 2016). This paper offers a subjective investigation of determined issues that damage or fix working environment connections.
Reason
The need of this investigation originates from the need to comprehend the social contrasts that should be comprehended when assessing worker execution (Shahwan, 2016). This understanding must originate from something other than the HR division, or the immediate supervisor. Consequently, assessing and understanding social contrasts and how representative’s exhibition is influenced by these distinctions is important to incorporate into work execution assessments (Shahwan, 2016). The creators demonstrate the holes in research encompassing the social varieties and their impacts on worker execution. This absence of comprehension from representatives to associations’ CEO can contrarily effect work execution anyplace on the range between brought work execution down to outrageous worker disappointment (Shahwan, 2016).
Research Questions
The creator came the exploration inquiries down the accompanying: (1) what social measurement Hofstede proposed, affected execution and efficiency at work (2) if any effect was discovered, how much impact the social measurement has on working environment execution (Shahwan, 2016, p.50).
Arrangement
Arrangement between the theme, proposal, reason, writing audit, and research questions was accomplished with this investigation as the writer remained on subject and tended to the proposition and research inquiries all through the paper.
Article Two
Goodstein, J., Butterfield, K., and Neale, N. (2016). Good Repair in the Workplace: A
Subjective Investigation and Inductive Model.
Theme
The experience of losing trust is a multi-dimensional encounter. It very well may be felt by those the individuals who have executed or by those whom are experience the less than desirable finish of a wrong-doing and end up loosing trust in another person. The endeavors taken by the guilty party and the reaction evoked in the getting party, is alluded to an “ethical fix”, Goodstien and Butterfield have made an investigation because of what strategies utilized by wrongdoers work and on the off chance that they are influenced by the thought process, strategy for conciliatory sentiment, or both.
Issue
This investigation is meaning on tending to the issue of good fix following ruptures of trust. The article centers around the techniques and reactions to the strategies utilized by the wrongdoers to recapture trust. The creators of this investigation were looking for comprehension “moral fix” which involved understanding the statement of regret technique, intention, and what blend of history, strategy, and rationale brings about what sorts of reaction. This investigation looks for a superior comprehend of the stuff to fix a relationship after hurtful occasions that have happened and what should be possible to rescue the relationship.
Reason
This investigation expresses what propels the guilty party to scan for reclamation, what moves they make, and why looking for recovery is significant. This examination is useful in the good and social domain because of its effect on how one may apologize and how one may get and statement of regret. Why connections have been damaged, guilty parties that care about the relationship, to recover confidence and respect that has been lost (Goodstein and Butterfield, 2015).
Research Questions
“(1) What components rouse wrongdoers to offer some kind of reparation with those they have hurt? (2) In what ways do guilty parties endeavor to offer some kind of reparation? (3) What results rise up out of endeavors to offer some kind of reparation?” (Goodstein and Butterfield, 2015, pg. 1, para 1).
Article Three
The third and last article being examined is Hartung and Wilson’s 2016 article titled “Conversational Moves That Matter”.
Theme
Hartung and Wilson are covering the theme of “learning discussions” which they clarify as discourse that at the same time covers investigation and assessment while reframing a person’s preparing by influencing their (1) thinking (2) suspicions (3) and point of view (Garmston and Wellman, 2009; Power, 2013; Senge, 1994).
Issue
The learning discussions are significant and gainful to the learning procedure; explicitly, in a working environment or scholarly based setting. The article is tending to the absence of information about what sorts of connections during the learning discussions were prominent; if any were outstanding, where the remarkable collaborations might they be able to be ordered by learning result.
Reason
The reason for the examination is to decide the usefulness of learning discussions in the general learning process, yet more explicitly about sorting any reoccurring learning results as to decide the viability of certain conversational moves (Hartung and Wilson, 2016). The investigation was custom fitted towards the working environment or scholastic setting and underlined the helpfulness of learning discussions in moving a perspective and how to bridle the most valuable conversational moves during learning discussions.
Research Questions
In prior research, Hartung and Wilson plot five explicit learning results, from which they likewise based their investigation in this flow study being analyzed. Utilizing these equivalent five results, the creators formed their examination and strategies. Hartung and Wilson (2016)
Research Question 1: What are the sorts and disseminations of conversational moves that happen in the conversational minutes wherein detailed learning results can be found?
Research Question 2: What, assuming any, conversational moves are altogether connected with various sorts of detailed learning results? (pg 257, para. 4).