HacDC CRISPR

Project Description

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

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). Media:https://wiki.hacdc.org/index.php/File:Jiang_W_Nat_Biotechnol_2013.pdf

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

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. We've contacted the vendor of the The ODIN kit regarding puchasing more ingredients rather than the whole kit; but we received no reply. We also contacted the Court Lab in Bethesda, where the HME63 strain was developed. They do not provide CRISPR plasmids or Cas9 but pointed us towards the gene we want for streptimycin resistance. CRISPR#2 v Control:https://wiki.hacdc.org/index.php/File:IMG_20160907_220305.jpg

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.