RfxCas13d Guide Cloning Protocol (v1.0 03-01-2023)


Required reagents (not included in starter kit)
	T4 DNA ligase 10xBuffer (incl ATP) (NEB #B0202S)
	T4 PNK enzyme (NEB #M0201S)
	ZYMO Research Mix & Go! E.coli Transformation Kit (#T3001)
	T7 DNA ligase (Enzymatics #L6020L)
	Rapid Ligase Buffer (Enzymatics #B1010)



Step 1 - Oligo annealing

	For each guide RNA you have ordered 2 oligos with 5' overhangs as depicted below. 
	The top oligo represents the reverse complement of your target site (equals the gRNA derived from http://cas13design.nygenome.org). 
	Make sure, top and bottom oligos contain matching overlaps to the BsmBI/Esp3I cut-sites in the guide backbone.
	Note, the insert can be a single guide RNA or a CRISPR array with multiple guide RNAs. 

	Overhang requirements for pLentiRNAGuide_003:

		    backbone				insert				       backbone
	Top 	    ggggtttg-3'		5’-AAACNNNNNNNNNNNNNNNNNNNNNNN-3’		5'-caagtaaacccc
	Bottom	    ccccaaactttg-5'    	    3’-NNNNNNNNNNNNNNNNNNNNNNNGTTC-5’		    3'-atttgggg

	Take each guide (top + bottom oligonucleotide pair; 100µM each) and mix them at equal ratios.

	- Annealing + 5'-phosphorylation reaction:
		Component					1x
		Top oligo [100µM]				1µl
		Bottom oligo [100µM]				1µl
		T4 DNA ligase 10xBuffer (incl ATP)		1µl
		T4 PNK enzyme (U/µl)				0.5µl
		H2O						6.5µl
		total						10µl

	- Incubate:
		30min	37°C
		5min	95°C
		Cool down to 4°C with 0.2°C/sec ramp rate
                                    
	- Dilute annealed primer mix 1:50 (like: 2µl + 98µl H2O)

	(Note, the undiluted annealing reaction can be stored at 4°C for at least 7 days.
	Prolonged storage or freezing cycles may reduce reactivity due loss of to 5'-phosphorylation.)



Step 2 - Ligation of annealed guide oligos, transformation and plasmid isolation.

	Warm up LB+Amp plates in 37°C for 1 h.
	Thaw ZYMO mix and go cells (stabl3) (10µl per vial) on ice for ~20 minutess before transformation.
	(We use stabl3 cells from Zymo, which are ‘mix and go’. Requires pre-warmed LB-Amp plates, and Amp-resistance. No heat-shock or outgrowth needed).
	Tips:
		a. Ligation and transformation reactions can be done efficiently using a plate-based layout with multichannel pipetting.
		b. You can distribute multiple transformation reaction on a single LB-Amp agar plate.

	- Ligation reaction:
		Component					1x
		Diluted and annealed dsDNA oligonucleotides	2 µl
		2x RLB buffer 					5 µl
		BsmBI-digested vector (~20ng/µl)		1 µl
		T7 DNA ligase (Enzymatics)			0.25 µl
		H2O						1.75 µl
		Total						10 µl

	- Set up a Mastermix containing H2O, RLB, and T7 DNA ligase (7µl)
	- Distribute 7µl into PCR tubes or 96 well plates. 
	- Add 2µl of the respective annealed oligo mix 1:100 dilution to each well.
	- Add 1µl of a provided BsmBI-digested pLentiRNAGuide_003 plasmid to each well (Each plasmid carries a unique bcgRNA).
	- Spin down briefly.
	- Pipette up and down 10x.
	- Incubate ~15 minutes at room temperature.
	- For transformation, add 2 µl ligation reaction to the competent cells, swirl gently, and incubate on ice for ~5 mins for fast transformation.
	- Distribute transformation reaction on on pre-warmed AMP agar plates
	- Incubate in 37°C for approximately 16 hours.

	Suggested negative controls:
	a.	Cells only (should yield no colonies)
	b.	Cells plus BsmBI-gigested vector (should yield zero or close to zero colonies)
	c.	Cells plus mock ligation (ligation reaction w/o annealed oligonucleotides) (usually yields a 0-20 colonies)
	Successful cloning is indicated by observing higher yield compared to control c.
	
	- Store LB-Amp agar plates at 4°C
	- Grow 1-2 mini cultures per construct and incubate in 37°C and 225 rotations per minute for approximately 16 hours.
	- Perform Mini Plasmid preparation

	Usually, we observe a ~80-90% cloning success rate. 
	Succuesful cloning will remove BsmBI-restriction sites, which may be used as an indicator for a control digestion (optional). 
	Plasmid concentrations typically range between 400-800 ng/µl.

	Plasmids can be normalized in concentration and pooled to create a plasmid pool for pooled virus production.