CRISPR/Cas9-mediated genome editing - Revision history

J Newitt: /* Golden gate assembly of annealed gRNA into pCRISPomyces-2 */

2023-11-16T10:02:10Z

Golden gate assembly of annealed gRNA into pCRISPomyces-2

← Older revision		Revision as of 10:02, 16 November 2023
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	==Golden gate assembly of annealed gRNA into [[pCRISPomyces-2]]==		==Golden gate assembly of annealed gRNA into [[pCRISPomyces-2]]==

	1. Set up 20 μl reactions with:		Set up 20 μl reactions with:

	• 100 ng backbone		• 100 ng backbone
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	• dH2O		• dH2O

	2. Incubate using golden gate programme (as below)		Incubate using golden gate programme (as below)

	• 10 ~~cycles of the following:~~		1. 10 minutes at 37°C

	• 10 minutes at ~~37°C~~		2. 10 minutes at 16°C

	~~• 10 minutes at 16°C~~		3. Go to step 1, 9 times

	• 5 minutes at 50°C		4. 1 cycle of 5 minutes at 50°C

	• 20 minutes at 65°C		5. 1 cycle of 20 minutes at 65°C

	• 4°C hold		6. 4°C hold

	==Transform Golden Gate plasmid into ''E. coli'' using heat shock==		==Transform Golden Gate plasmid into ''E. coli'' using heat shock==

Sam Prudence: /* Conjugate ET into Streptomyces treat with antibiotics to select for ex-conjugants */

2019-11-14T16:52:26Z

Conjugate ET into Streptomyces treat with antibiotics to select for ex-conjugants

← Older revision		Revision as of 16:52, 14 November 2019
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	4. Pre-germinate Streptomyces spores in [[2X YT]] at 50°C for 10 minutes, leave to cool		4. Pre-germinate Streptomyces spores in [[2X YT]] at 50°C for 10 minutes, leave to cool

	5. Combine cells, centrifuge and resuspended in 200 ul [[LB]]		5. Combine cells, centrifuge and resuspended in 200 μl [[LB]]

	6. Plate dilutions of approx. 200 ul onto [[SFM]] agar + MgCl2 and incubated at 30°C for 16-20 hours (I increase the time for CRISPR conjugations compared to other vectors e.g. integrative plasmids i.e. use 20 hours rather than 16)		6. Plate dilutions of approx. 200 μl onto [[SFM]] agar + MgCl2 and incubated at 30°C for 16-20 hours (I increase the time for CRISPR conjugations compared to other vectors e.g. integrative plasmids i.e. use 20 hours rather than 16)

	7. Overlay with 1 ml of dH2O + 0.5 mg Nalidixic acid + 1.25 mg Apramycin to selectively kill the ''E. coli''		7. Overlay with 1 ml of dH2O + 0.5 mg Nalidixic acid + 1.25 mg Apramycin to selectively kill the ''E. coli''

Sam Prudence: /* Transform finished cosmid into ET cells using electroporation */

2019-11-14T16:51:04Z

Transform finished cosmid into ET cells using electroporation

← Older revision		Revision as of 16:51, 14 November 2019
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	7. Recover plasmid by same method as previously		7. Recover plasmid by same method as previously

	==Transform finished ~~cosmid~~ into ET cells using electroporation==		==Transform finished plasmid into ET cells using electroporation==
	1. Streptomyces strains contain a methyl-sensing restriction system therefore disrupted cosmids must initially be passaged through a non-methylating ''E. coli'' strain		1. Streptomyces strains contain a methyl-sensing restriction system therefore disrupted cosmids must initially be passaged through a non-methylating ''E. coli'' strain

	2. Thaw ET cells on ice		2. Thaw ET cells on ice

	3. Cool cuvette		3. Cool cuvette on ice

	4. ~~Take to electroporation machine – set to Bacteria, EC2 (up)~~		4. Electropotate using appropriate settings using 1 μl cosmid DNA in 50 μl of electrocompetent ET12567 cells containing the driver plasmid [[pUZ8002]] and “pulse”

	5. ~~Put 1 μl cosmid DNA into 50 μl~~ of ~~electrocompetent ET12567 cells containing~~ the ~~driver plasmid [[pUZ8002]] and “pulse”~~		5. Take care not to hold the metal parts of the cuvette to warm it up

	6. ~~Take care not to hold the metal parts of the cuvette to warm it up~~		6. Reading should be around 5.4

	7. ~~Reading should be around 5.4~~		7. Add 900 μl [[LB]] and pipette tip mix

	8. ~~Add 900 μl [[LB]] and pipette tip mix~~		8. Transfer to eppendorf & grow at 37ºC up for 1 hour to recover

	~~9. Transfer to microcentrifuge~~		11. Plate onto [[LB]] agar containing kanamycin, apramycin and chloramphenicol to select for the incoming plasmid

	~~10. Grow up for 1 hour to recover~~

	11. Plate onto [[LB]] agar containing kanamycin, apramycin and chloramphenicol to select for the incoming ~~cosmid~~

	12. Incubate plates at 37ºC overnight		12. Incubate plates at 37ºC overnight

Sam Prudence: /* Select for successful transformants using blue/white selection */

2019-11-14T16:46:07Z

Select for successful transformants using blue/white selection

← Older revision		Revision as of 16:46, 14 November 2019
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	==Select for successful transformants using blue/white selection==		==Select for successful transformants using blue/white selection==

	Golden gate cloning into [pCRISPomyces-2] uses blue white screening to select for successful transformants (See AddGene blog [https://blog.addgene.org/plasmids-101-blue-white-screening here] for an explaination of blue-white screening). Pick white colonies and grow overnight in 10 ml [[LB]] + Apramycin then extract plasmid DNA from successful colony(s).		Golden gate cloning into [[pCRISPomyces-2]] uses blue white screening to select for successful transformants (See AddGene blog [https://blog.addgene.org/plasmids-101-blue-white-screening here] for an explaination of blue-white screening). Pick white colonies and grow overnight in 10 ml [[LB]] + Apramycin then extract plasmid DNA from successful colony(s).

	2. Grow up successful (white) colonies in [[LB]] at 37°C overnight		2. Grow up successful (white) colonies in [[LB]] at 37°C overnight
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	9. Discard the flow through and elute plasmid DNA in 50 μl dH2O		9. Discard the flow through and elute plasmid DNA in 50 μl dH2O

	10. Quantify using Nanodrop and take note of concentration for use in ~~golden gate reaction~~		10. Quantify using Nanodrop and take note of concentration for use in the next steps

	==Linearize gRNA vector using XbaI digestion==		==Linearize gRNA vector using XbaI digestion==

Sam Prudence: /* Select for successful transformants using blue/white selection */

2019-11-14T16:44:54Z

Select for successful transformants using blue/white selection

← Older revision		Revision as of 16:44, 14 November 2019
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	==Select for successful transformants using blue/white selection==		==Select for successful transformants using blue/white selection==

	1. Pick white colonies and grow overnight in 10 ml [[LB]] + Apramycin~~. Extract~~ DNA from successful colony(s) ~~(plasmid prep)~~		Golden gate cloning into [pCRISPomyces-2] uses blue white screening to select for successful transformants (See AddGene blog [https://blog.addgene.org/plasmids-101-blue-white-screening here] for an explaination of blue-white screening). Pick white colonies and grow overnight in 10 ml [[LB]] + Apramycin then extract plasmid DNA from successful colony(s).

	2. Grow up successful colonies in [[LB]] at 37°C overnight		2. Grow up successful (white) colonies in [[LB]] at 37°C overnight

	3. Recover plasmid using the QIAprep Spin Miniprep kit (Qiagen)		3. Recover plasmid using the QIAprep Spin Miniprep kit (Qiagen) (or a plasmid miniprep kit of choice)

	4. Pellet ~3 ml cells by centrifugation		4. Pellet ~3 ml cells by centrifugation

Sam Prudence: /* Anneal gRNA using oligo spacer programme */

2019-11-14T16:37:28Z

Anneal gRNA using oligo spacer programme

← Older revision		Revision as of 16:37, 14 November 2019
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	==Anneal gRNA using oligo spacer programme==		==Anneal gRNA using oligo spacer programme==

	1. Mix 5 μl Forward gRNA and 5 μl Reverse gRNA oligo ~~(100μM)~~ with 90 μl HEPES buffer		1. Mix 5 μl 100μM Forward gRNA and 5 μl 100μM Reverse gRNA oligo with 90 μl HEPES buffer

	2. Heat to 95°C in a thermocycler and then ramping down to 4°C at 0.1°C per second using spacer oligos programme		2. Heat to 95°C in a thermocycler and then ramping down to 4°C at 0.1°C per second using spacer oligos programme

Sam Prudence: /* Anneal gRNA using oligo spacer programme */

2019-11-14T16:36:51Z

Anneal gRNA using oligo spacer programme

← Older revision		Revision as of 16:36, 14 November 2019
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	==Anneal gRNA using oligo spacer programme==		==Anneal gRNA using oligo spacer programme==

	1. Mix 5 μl Forward gRNA and 5 μl Reverse gRNA with 90 μl HEPES buffer		1. Mix 5 μl Forward gRNA and 5 μl Reverse gRNA oligo (100μM) with 90 μl HEPES buffer

	2. Heat to 95°C in a thermocycler and then ramping down to 4°C at 0.1°C per second using spacer oligos programme		2. Heat to 95°C in a thermocycler and then ramping down to 4°C at 0.1°C per second using spacer oligos programme

Sam Prudence: /* Designing gRNA */

2019-11-14T16:33:22Z

Designing gRNA

← Older revision		Revision as of 16:33, 14 November 2019
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	4. Design ~20 NT gRNA with a Tm > 60°C		4. Design ~20 NT gRNA with a Tm > 60°C

	5. Blast ALL 4 POSSIBLE gRNAs (I.E where N is either A,C,T, or G) against full genome to check gRNA specificity. ~~More~~ important to not have matching on 3’ end		5. Blast ALL 4 POSSIBLE gRNAs (I.E where N is either A,C,T, or G) against the full genome to check gRNA specificity. It is more important to not have matching sequences on the 3’ end

	6. Add BbsI cut site sticky ends for golden gate assembly		6. Add BbsI cut site sticky ends for golden gate assembly

Sam Prudence: /* Designing gRNA */

2019-11-14T16:32:29Z

Designing gRNA

← Older revision		Revision as of 16:32, 14 November 2019
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	6. Add BbsI cut site sticky ends for golden gate assembly		6. Add BbsI cut site sticky ends for golden gate assembly

	The ~~program [~~[https://crispy.secondarymetabolites.org/#/input CRISPY-web]] can also be used to gRNA design, simply upload your target genome and define a target region. The software will then provide a list the most unique PAM sequences & gRNAs it can find within the target region.		The website [https://crispy.secondarymetabolites.org/#/input CRISPY-web] can also be used to gRNA design, simply upload your target genome and define a target region. The software will then provide a list the most unique PAM sequences & gRNAs it can find within the target region.

	==Anneal gRNA using oligo spacer programme==		==Anneal gRNA using oligo spacer programme==

Sam Prudence: /* Designing gRNA */

2019-11-14T16:32:07Z

Designing gRNA

← Older revision		Revision as of 16:32, 14 November 2019
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	==Designing gRNA==		==Designing gRNA==

	1. Open new copy of gene sequence		1. Open a new (unedited) copy of the target gene sequence

	2. Look at REVERSE STRAND		2. Look at the REVERSE STRAND

	3. Find NGG (PAM) within gene to be removed (search for ANGG or better TANGG to find unique sequences in high GC genomes)		3. Find NGG (PAM) within gene to be removed (search for ANGG or better TANGG to find unique sequences in high GC genomes)
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	6. Add BbsI cut site sticky ends for golden gate assembly		6. Add BbsI cut site sticky ends for golden gate assembly

			The program [[https://crispy.secondarymetabolites.org/#/input CRISPY-web]] can also be used to gRNA design, simply upload your target genome and define a target region. The software will then provide a list the most unique PAM sequences & gRNAs it can find within the target region.

	==Anneal gRNA using oligo spacer programme==		==Anneal gRNA using oligo spacer programme==