Restructered pre-import
This commit is contained in:
Liz Cray
234
Old Pages/Old Wiki/DC_CRISPR_Initiative_at_HacDC.md
Executable file
234
Old Pages/Old Wiki/DC_CRISPR_Initiative_at_HacDC.md
Executable file
@@ -0,0 +1,234 @@
|
||||
## Project Description
|
||||
|
||||
- Organizers: Enrique C., Nancy C. Wolfson, Bobby B.
|
||||
- Contact: crispr@hacdc.org
|
||||
|
||||
CRISPR-Cas9 is a groundbreaking new (2012 vintage) gene editing
|
||||
technique. While gene editing is not a new concept, previous methods
|
||||
were far more expensive, slow and restricted in capabilities than
|
||||
CRISPR. Further, whereas previous methods only successfully edited a few
|
||||
percent of the exposed cells, CRISPR's efficiency approaches 100%. This
|
||||
is particularly important for gene editing in living multi-cellular
|
||||
organisms. The new technique is dramatically accelerating the pace of
|
||||
genetic engineering since its invention in 2012.
|
||||
|
||||
CRISPR is an acronym that describes a genetic curiosity observed several
|
||||
decades ago: Clusters of Regularly Interspaced Short Palindromic
|
||||
Repeats. A few years go it was recognized that these odd DNA sequences
|
||||
in bacteria are deactivated virus DNA and make up a kind of Virus
|
||||
Definition Database. Finally, it was discovered that a protein exists
|
||||
which goes around scanning bacteria DNA searching for matches to virus
|
||||
DNA sequences and, when a match is found, cuts the gene in the bacteria
|
||||
DNA very precisely and efficiently. This protein was termed Cas9. In
|
||||
effect, bacteria and other organisms already have an excellent built-in
|
||||
gene-editing mechanism, and scientists have since learned to hijack that
|
||||
mechanism using engineered RNA sequences that direct the Cas9 protein to
|
||||
cut DNA in any desired location in the genome.
|
||||
|
||||
The Cas9 mechanism only cuts the DNA in one location, leaving DNA repair
|
||||
mechanisms to fix the double-strand cut. Repair mechanisms for such a
|
||||
serious double-strand break are so imperfect as to incapacitate the gene
|
||||
with errors most of the time. Thus the Cas9 protein deactivates genes
|
||||
rather than removing them. However, by programming two custom RNA target
|
||||
strands, two Cas9 proteins can be used in tandem to excise part of a
|
||||
genome altogether. To add a new gene, there must be enough of that gene
|
||||
floating around during the CRISPR process that it becomes incorporated
|
||||
into the genome via the repair mechanisms.
|
||||
|
||||
These are the best videos we've found so far: Genome Editing with
|
||||
CRISPR-Cas9 by McGovern Institute for Brain Research
|
||||
<https://www.youtube.com/watch?v=2pp17E4E-O8>
|
||||
|
||||
What is CRISPR? by Bozeman Science
|
||||
<https://www.youtube.com/watch?v=MnYppmstxIs>
|
||||
|
||||
Read more here: <https://en.wikipedia.org/wiki/CRISPR>
|
||||
|
||||
Read more here: <https://en.wikipedia.org/wiki/CRISPR>
|
||||
|
||||
In the ODIN kit experiment, bacteria (E. coli HME63 strain) are modified
|
||||
to add resistance to the antibiotic streptomycin. The kit provides the
|
||||
vulnerable bacteria, the resistance gene, and growth media with and
|
||||
without antibiotic. The original unmodified bacteria can only grow on
|
||||
the plain agar media whereas bacteria with a successfully edited genome
|
||||
will also grow on the streptomycin-laced agar. This is similar to Wenyan
|
||||
Jiang, David Bikard, David Cox, Feng Zhang and Luciano Marraffini,
|
||||
RNA-guided editing of bacterial genomes using CRISPR-Cas systems, Nature
|
||||
Biotechnology 31(3), pp.233 (2013).
|
||||
<http://zlab.mit.edu/assets/reprints/Jiang_W_Nat_Biotechnol_2013.pdf>
|
||||
|
||||
## Financial Support / Sponsors
|
||||
|
||||
HacDC - 8/2016 - purchased Bacterial DIY CRISPR kit. Enrique C -
|
||||
11/2016- purchased Bacterial DIY CRISPR Kit. Nancy W - purchased
|
||||
Bacterial DIY CRISPR kit. Enrique C - 5/2017 - purchased Bacterial DIY
|
||||
CRISPR Kit Refill. Nancy W - 2017 - purchased Bacterial DIY CRISPR kit
|
||||
refill. Nancy W - 2017 - purchased temperature-controlled water bath.
|
||||
Nancy W - 2017 - loaned 1600X Optical Microscope with USB camera.
|
||||
Enrique C - 2017 - purchased supplies and gram staining kit. The ODIN -
|
||||
01/2018- provided free sample of next-gen CRISPR kit.
|
||||
|
||||
## Activities and Goals
|
||||
|
||||
DC CRISPR Initiative is our effort to learn about, perform, and teach
|
||||
CRISPR genetic editing at HacDC. To begin the project, we???ve ordered a
|
||||
Do-It-Yourself CRISPR Kit, which includes (supposedly) all the tools and
|
||||
ingredients needed to perform a CRISPR procedure a few times. We???ll
|
||||
hold a few events at HacDC to go through the procedure and document our
|
||||
experience. Eventually we???ll create a guide that older high school
|
||||
kids can follow. This project also explores interest in molecular
|
||||
biology and genetics at HacDC. We're just starting! Keep an eye out for
|
||||
CRISPR events in our MeetUp page, on the mailing list, and our Blabber
|
||||
discussion forum.
|
||||
|
||||
## Project Team Members
|
||||
|
||||
Enrique C. - Project Manager and Point of Contact Nancy W. - Project
|
||||
Development Lead Bobby B. - Molecular Biology Advisor Sophia M. -
|
||||
Scientific Advisor
|
||||
|
||||
## Worklog
|
||||
|
||||
July 30, 2016 We received the CRISPR kit purchased with Project
|
||||
EXPANSION funds (thanks!).
|
||||
|
||||
August 2, 2016 Nancy and Enrique inventoried the ODIN kit and designated
|
||||
the small classroom fridge as the "NO FOOD" Project CRISPR fridge.
|
||||
|
||||
August 5, 2016 Nancy and Enrique prepared four Petri dishes (two plain
|
||||
agar, two streptomycin-agar). The agar and
|
||||
antibiotic(streptomycin)-laced agar are gel-like substances similar to
|
||||
gelatin. They come as powders which must be mixed with water and heated
|
||||
to dissolve. The recipe is proportioned for seven Petri dishes but we
|
||||
scaled down to one of each, scaling the agar powders and water by
|
||||
one-seventh. Even so we were able to coat two dishes with each growth
|
||||
medium. We didn't have distilled or deionized water and used bottled
|
||||
purified drinking water in a pinch. The mixture (agar gel only, no
|
||||
bacteria!) was heated in the microwave 7 seconds at a time. It took 4-5
|
||||
cycles until the powders were fully dissolved and the liquid
|
||||
transparent, then another 5 minutes until they were cool enough to
|
||||
handle and pour into the plastic Petri dishes. The dishes cooled at room
|
||||
temperature for an hour to remove some condensation (the covered hot
|
||||
liquid creates condensation on the lid), then placed in the fridge. Two
|
||||
are agar (no antibiotic) and two are streptomycin/Kan agar (antibiotic
|
||||
laced).
|
||||
|
||||
August 10, 2016 Ken, Bobby, Nancy, and Enrique. We streaked some of the
|
||||
original E. coli HME63 bacteria onto two plain agar plates. Plate 1 was
|
||||
left out tonight (the bacteria need to grow). Plate 2 was immediately
|
||||
refrigerated and will be taken out to grow just before the actual
|
||||
experiment.
|
||||
|
||||
August, 2016 Nancy, Ken and Enrique. We performed the CRISPR experiment,
|
||||
but realized we don't have a constant-temperature water bath. We used an
|
||||
IR thermometer and the microwave to prepare and maintain a water bath of
|
||||
approximately 42C. The incubation period post-CRISPR is also quite long,
|
||||
up to 4 hours at room temperature, which makes this experiment
|
||||
problematic as an after-work evening activity. It'd work better on a
|
||||
weekend day or holiday since we have day jobs.
|
||||
|
||||
September, 2016 Nancy, Enrique. The first CRISPR-modified bacteria on
|
||||
the Strep-Kan plate was left at room temperature over the weekend (48
|
||||
hours). However, no bacteria colonies could be easily seen in the plate.
|
||||
The plate was left at room temperature several more days with no change.
|
||||
It looks like our first attempt did not wildly succeed.
|
||||
|
||||
September, 2016 Enrique. I prepared a new set of Agar (3) and Strep-Kan
|
||||
Agar (3) plates for troubleshooting experiments and a second try at
|
||||
CRISPR. We should test the full CRISPR protocol again, both on Agar and
|
||||
Strep-Kan plates, but also test the survival of bacteria at several
|
||||
other steps: Transformation Mix only and Transformation Mix + tracrRNA +
|
||||
crRNA.
|
||||
|
||||
September, 2016 Enrique and Nancy. We developed a troubleshooting
|
||||
protocol under the suspicion that maybe none of the bacteria are
|
||||
surviving (we did not make a plain Agar control plate last time). We
|
||||
also tested the 'sterile' innoculation loops vs. a bag of zip-ties we
|
||||
found laying around. Nancy brought an alcohol thermometer that's
|
||||
probably more accurate in water than the IR thermometer. The resulting
|
||||
plates were incubated for 24-48 hours. It was difficult to re-suspend
|
||||
the bacteria in solution after harvesting from the first plate. A vortex
|
||||
generator (basically a strong vibrator) would be helpful.
|
||||
|
||||
September, 2016 Enrique and Nancy. Hm... there are some specs on the
|
||||
CRISPR plate this time, just 2-3 colonies though. All the other (Agar)
|
||||
plates had tons of bacteria, so clearly the low efficiency is at the
|
||||
CRISPR step with the Template DNA. Also we brought in more Bleach for
|
||||
disposal of old samples, and bleach wipes.
|
||||
|
||||
September, 2016 Enrique. I located a much better (real) biological
|
||||
microscope with up to 1000x magnification in the basement. Man our
|
||||
inventory sucks; I had no idea we had this thing. Images are much
|
||||
better, although I'm not certain we're looking at individual bacteria. I
|
||||
also purchased a hotplate so we can actually control temperature baths
|
||||
in the future.
|
||||
|
||||
January, 2017 See notebook.
|
||||
|
||||
February, 2017. The third trial of the CRISPR process gave us a positive
|
||||
control sample, which makes the result inconclusive. While the Agar
|
||||
media grew more, both the CRISPR and original LD 21 bacteria grew
|
||||
somewhat on the Strep-Kan media. We perfomed a second control
|
||||
experiment, plating 100uL of the LD21 bacteria and the
|
||||
Transformation-Mix HME 63 strain with 400uL of DI water each, onto
|
||||
Strep-Kan media.
|
||||
|
||||
June, 2017 Purchased Bacterial CRISPR refill kit using HacDC Project
|
||||
CRISPR funds.
|
||||
|
||||
July 2017 - Bobby, Nancy, Enrique, Sophia and Richard. Met to plan
|
||||
workshop. Bobby gave us his Introduction to Molecular Biology talk - it
|
||||
was about 90 minutes but could probably be shortened to 60 if needed.
|
||||
Prepared fresh Agar and Strep-Kan plates. Sophia showed us a new
|
||||
(correct) streaking technique. We plated some bacteria but they overgrew
|
||||
(storage place on top of the fridge is too warm in the summer), making
|
||||
it impossible to isolated a colony. Sophia took a second batch home and
|
||||
it was better with some isolated colonies.
|
||||
|
||||
August 23, 2017 - Sophia, Nancy and Enrique. Trial 4 of the full CRISPR
|
||||
process. Enrique took the resulting plates home to monitor growth at
|
||||
room temperature. No growth observed on the CRISPR sample. Control
|
||||
sample grew quickly.
|
||||
|
||||
December 11, 2017 - Nancy and Enrique. Contacted kit supplier to obtain
|
||||
fresh ingredients.
|
||||
|
||||
January 09, 2017 - Enrique. Prepared fresh Agar and Strep-Kan Agar
|
||||
plates. Checked for contamination growth 48 hours (none).
|
||||
|
||||
January 12, 2017 - Nancy, Enrique and guests Tobi and Tom. Trial 4.5 of
|
||||
the CRISPR process. Used bacteria straight from the supply rather than a
|
||||
colony (not available). The result after 48 hours was much growth on the
|
||||
Agar plate and zero growth on the Strep-Kan plate. (no modification)
|
||||
|
||||
## Sample Inventory
|
||||
|
||||
**CRISPR Trial 8/24/2017** LB-Agar Plate at 1 and 20 Hours:
|
||||
|
||||
|
||||
|
||||
StrepKan Plate at 1, 20, 28 and 57 Hours:
|
||||
|
||||
|
||||
|
||||
|
||||
|
||||
**CRISPR Trial 1/12/2018**Clean Agar plate after 48 hours incubation. No
|
||||
contamination growth observed.
|
||||
[Media:CRISPR-180112-Agar_blank_48h.jpeg](Media:CRISPR-180112-Agar_blank_48h.jpeg "wikilink")
|
||||
Clean Strep-Kan plate after 48 hours incubation. No contamination growth
|
||||
observed.
|
||||
[Media:CRISPR-180112-StrepKan_blank_48h.jpeg](Media:CRISPR-180112-StrepKan_blank_48h.jpeg "wikilink")
|
||||
Agar plate with the post-protocol bacteria incubated 48 hours (control
|
||||
sample): much growth (overgrown).
|
||||
[Media:CRISPR-180112-Agar_Bact_48h.jpeg](Media:CRISPR-180112-Agar_Bact_48h.jpeg "wikilink")
|
||||
Strep-Kan plate with the post-protocol bacteria incubation 48 hours
|
||||
(CRISPR result): no growth.
|
||||
[Media:CRISPR-180112-StrepKan_CrisprBact_48h.jpeg](Media:CRISPR-180112-StrepKan_CrisprBact_48h.jpeg "wikilink")
|
||||
|
||||
## News References
|
||||
|
||||
- Video: bacteria evolving antibiotic resistance in 11 days.
|
||||
<https://www.youtube.com/watch?v=yybsSqcB7mE>
|
||||
- Video: CRISPR Patent Controversy:
|
||||
<https://www.youtube.com/watch?v=IboHEQumDGc>
|
||||
Reference in New Issue
Block a user