HacDC CRISPR - Revision history

Summakor: asdf

2017-01-19T02:26:08Z

asdf

← Older revision		Revision as of 02:26, 19 January 2017
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	~~==Project Description==~~		CRISPR Initiative at HacDC page has moved to:

	CRISPR-Cas9 is a groundbreaking gene editing technique discovered and invented less than five years ago. While science fiction writers were imagining 'nanobots' that could be programmed to read and edit DNA, biologists discovered bacteria already have nanobots (Cas) that read and edit their DNA. Shortly thereafter, techniques were invented to reprogram, control and even modify these nanobots. While gene editing is not a new invention, 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.		https://wiki.hacdc.org/index.php/DC_CRISPR_Initiative_at_HacDC

	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 it was hypothesized that they are a Virus Definition Database. Finally, it was discovered that a protein exists which goes around scanning bacteria DNA searching for matches to these virus DNA sequences and, when a match is found, cuts the gene in the bacteria DNA very precisely and efficiently. This protein was termed Cas for "CRISPR associated" protein. 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 purchased 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 & 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.
	~~[[File:https://wiki.hacdc.org/index.php/File:IMG_20160913_232219.jpg]]~~

	~~September 27, 2016~~
	Enrique. Prepared three new Agar plates and three new Step-Kan Agar plates. Purchased a hot plate, thermometer, 40 fresh petri dishes, 50g of plain Agar, a gram-negative staining kit for imaging E.coli, zip-lock bags and some other supplies.

Summakor: /* Worklog */ img

2016-09-28T04:41:18Z

Worklog: img

← Older revision		Revision as of 04:41, 28 September 2016
Line 58:		Line 58:
	September, 2016		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.		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.
	[[~~Media~~:https://wiki.hacdc.org/index.php/File:IMG_20160913_232219.jpg]]		[[File:https://wiki.hacdc.org/index.php/File:IMG_20160913_232219.jpg]]

	September 27, 2016		September 27, 2016
	Enrique. Prepared three new Agar plates and three new Step-Kan Agar plates. Purchased a hot plate, thermometer, 40 fresh petri dishes, 50g of plain Agar, a gram-negative staining kit for imaging E.coli, zip-lock bags and some other supplies.		Enrique. Prepared three new Agar plates and three new Step-Kan Agar plates. Purchased a hot plate, thermometer, 40 fresh petri dishes, 50g of plain Agar, a gram-negative staining kit for imaging E.coli, zip-lock bags and some other supplies.

Summakor: asdfd

2016-09-28T04:35:36Z

asdfd

← Older revision		Revision as of 04:35, 28 September 2016
Line 1:		Line 1:
	==Project Description==		==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-Cas9 is a groundbreaking gene editing technique discovered and invented less than five years ago. While science fiction writers were imagining 'nanobots' that could be programmed to read and edit DNA, biologists discovered bacteria already have nanobots (Cas) that read and edit their DNA. Shortly thereafter, techniques were invented to reprogram, control and even modify these nanobots. While gene editing is not a new invention, 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~~.		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 it was hypothesized that they are a Virus Definition Database. Finally, it was discovered that a protein exists which goes around scanning bacteria DNA searching for matches to these virus DNA sequences and, when a match is found, cuts the gene in the bacteria DNA very precisely and efficiently. This protein was termed Cas for "CRISPR associated" protein. 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.

	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.


Line 22:		Line 20:

	==Activities and Goals==		==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.		DC CRISPR Initiative is our effort to learn about, perform, and teach CRISPR genetic editing at HacDC. To begin the project, we’ve purchased 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==		==Project Team Members==
Line 61:		Line 59:
	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.		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.
	[[Media:https://wiki.hacdc.org/index.php/File:IMG_20160913_232219.jpg]]		[[Media:https://wiki.hacdc.org/index.php/File:IMG_20160913_232219.jpg]]

			September 27, 2016
			Enrique. Prepared three new Agar plates and three new Step-Kan Agar plates. Purchased a hot plate, thermometer, 40 fresh petri dishes, 50g of plain Agar, a gram-negative staining kit for imaging E.coli, zip-lock bags and some other supplies.

Summakor: /* Worklog */ microscope photo

2016-09-26T03:20:35Z

Worklog: microscope photo

← Older revision		Revision as of 03:20, 26 September 2016
Line 56:		Line 56:
	September, 2016		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.		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]]		[[CRISPR & Control:https://wiki.hacdc.org/index.php/File:IMG_20160907_220305.jpg]]

	September, 2016		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.		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.
			[[Media:https://wiki.hacdc.org/index.php/File:IMG_20160913_232219.jpg]]

Summakor: /* Worklog */ photo

2016-09-26T03:14:34Z

Worklog: photo

← Older revision		Revision as of 03:14, 26 September 2016
Line 56:		Line 56:
	September, 2016		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.		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		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.		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.

Summakor: /* Project Description */ paper

2016-09-26T03:11:44Z

Project Description: paper

← Older revision		Revision as of 03:11, 26 September 2016
Line 19:		Line 19:

	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).		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==		==Activities and Goals==

Summakor: /* Worklog */ court lab

2016-09-26T03:09:06Z

Worklog: court lab

← Older revision		Revision as of 03:09, 26 September 2016
Line 54:		Line 54:

	September, 2016		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.		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.

	September, 2016		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.		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.

Summakor: /* Worklog */ fff

2016-09-26T03:06:35Z

Worklog: fff

← Older revision		Revision as of 03:06, 26 September 2016
Line 40:		Line 40:
	August 10, 2016		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.		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.

Summakor: /* Project Description */ asdf

2016-09-26T02:43:02Z

Project Description: asdf

← Older revision		Revision as of 02:43, 26 September 2016
Line 1:		Line 1:
	==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==

Summakor: /* Activities and Goals */ sZDsSDF

2016-09-13T03:23:08Z

Activities and Goals: sZDsSDF

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	==Activities and Goals==		==Activities and Goals==
	~~HacDC Project~~ CRISPR 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.		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==		==Project Team Members==