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