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==Project Description==
==Project Description==


CRISPR-Cas9 is a groundbreaking new gene editing technique in molecular biology. In brief, ten years ago it was found that bacteria possess a self-defense mechanism against viruses that includes a virus definition database and a gene removal mechanism. The bacterial chromosomal DNA contains small pieces of the DNA sequences of harmful viruses. The defense mechanism reads the chromosomal DNA and compares it to the known virus sequence. If a match is found, the sequence is excised. This mechanism can therefore be exploited to excise any gene by replacing the virus definition sequences in the CRISPR sequence with the target sequence. Once the DNA is cut, a new gene can be inserted with reasonable success. The cost of this technique is dramatically reduced from pre-existing gene editing techniques.
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:
Read more here:
https://en.wikipedia.org/wiki/CRISPR
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.
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).


==Activities and Goals==
==Activities and Goals==

Revision as of 18:43, 25 September 2016

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).

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.