pSpCas9(BB)-2A-Puro (PX459) V2.0 (Plasmid #62988) cloning methods

[Sean Collier]

Hello,

I am relatively new at using the CRISPR system for my research. I am interested in doing knockout experiments in a mammalian cell line that I am using. Can use an alternative cloning method (i.e. infusion or use a SDM kit from NEB) to have my gRNA insert into this vector?

Best,

Sean

[Alex Brown]

I've talked with way too many people that were having trouble with this basic step, so I optimized it a bit. I use it for the pSPsgRNA plasmid from the Gersbach lab, but have also used it with the plasmid you describe (same sticky ends). I've handed this to undergrads with less than 2 weeks lab experience and they got loads of colonies. In my hands, hundreds of different sgRNA made, about 95% colonies correct. So if you have trouble... it's your reagents. I'd link the publication, but it's a book chapter that is taking forever to go to print. Good luck.

This protocol for construction of single-guide RNA expression vectors takes approximately 2 hours and assumes that the backbone

vector has been plasmid prepped and the oligos have been resuspended in water at 100uM each. The cleavage of the empty vector,

phosphorylation of the annealed oligos, and final product ligation reactions occur simultaneously in a single reaction, without need for

purification. Since there is no gel cleanup step, re-ligation of the dual-BbsI site oligo is possible. Ligation of the custom insert is favored

by maintaining a large molar excess (~20:1, insert:vector). Additionally, the old insert still contains BbsI sites, so any undigested or

reassembled empty vector is steadily eliminated through multiple digestion-ligation cycles. In practice, 3 digestion-phosphorylation-ligation

cycles and the minimal Mix N' Go transformation protocol is normally sufficient to yield hundreds of colonies, of which, the vast

majority of colonies are positive for the desired construct.

Materials

› sgRNA plasmid (empty)

› U6 promoter, dual BbsI (BpiI) cloning sight for protospacer (target) sequence

› Custom designed, complementary oligonucleotides:

› Sense (+): 5'- CACC G {19-20 bases preceding PAM} -3'

› Antisense (-): 3'-AAAC {reverse complement/antiparallel of +} C -3'

› 1X Tris-buffered saline (TBS), pH ~7.4, 50mM Tris, 150mM NaCl - 49uL/sample

› 10X T4 DNA Ligase Buffer (FRESH ALIQUOT! avoid multiple freeze-thaws) - 2uL/sample

› BpiI restriction endonuclease (BbsI works, too, but must store in -80C) - 1uL/sample

› T4 Polynucleotide kinase (PNK) - 1uL/sample

› T4 DNA ligase - 1uL/sample

› Molecular Biology Grade Water

Procedure

1. Mix 0.5 μL of each 100 uM oligo with 49 μL of 1x TBS (1.0 μM ea. = ~15 ng/μL total DNA).

2. Anneal the oligos by incubating the 50 μL reaction at 95°C for 5 min, then 25°C for 3 min (QUIKMELT

program). Alternatively, use the longer ANNEAL or MELT programs.

3. Mix 1 μL of annealed and diluted oligos with 170 ng gRNA vector, 2 μL 10x T4 ligase buffer, 1 μL BpiI (BbsI), 1

μL T4 DNA ligase, and 1 μL T4 polynucleotide kinase (PNK) into 20 μL reactions. Each reaction should contain

<200 ng total DNA (plasmid and oligos) while maintaining an insert-to-vector ratio of ~20:1. Fresh T4 ligase

buffer (<20 freeze-thaws, on ice) should be used. This ensures that the DTT needed for PNK is not oxidized

and the ATP required for PNK/ligase activity is not degraded.

4. Incubate reaction mixture at 37°C for 5 min, followed by 10 min at 16°C. Repeat this step twice more for a total

of 3 cycles and holding at 16°C until ready to proceed to step 5 (DIG-LIG program).

5. Transform the assembled gRNA by mixing 1.5 μL of the reaction with ~10-20 μL of thawed Mix N’ Go

chemically competent cells. Incubate on ice for 5 minutes then add ~50-75 μL of pre-warmed SOC and recover

cells at 37°C while shaking for 15-30 min. Plate all cells onto pre-warmed LB+carb100 agar petri dishes and

incubate at 37°C overnight. You can use any chemically competent cells if you prefer traditional heat-shock transformation.

6. Optional: verify positive integration by colony PCR using M13F/antisense gRNA or sense gRNA/M13R oligo

[nikita modi]

I second Alex's protocol. the complete digestion/ligation is good. but a couple of things to note- use the T4 ligase vs the quick ligase. quick ligase has PEG which inhibits transformation. and aliquot your Bbs1 too.although it is good for over 10 freeze-thaw cycles 

On Tuesday, September 20, 2016 at 4:27:40 PM UTC-5, Sean Collier wrote:

[Alex Brown]

Great point about the ligase, Nikita, I often forget we all have subtle, yet important differences in our preferred enzymes and reagents. Case in point, we've opted for Thermo's BpiI in lieu of BbsI due to recommended storage conditions. All the others are from NEB. Also, we use so much that it is never more than a few months old.

On a related note, I've never optimized the quantities of enzymes used in this protocol and I suspect they are in gross excess of what is needed. You could probably halve the amounts and be just as good.

[Ben Steil]

How necessary is the addition of PNK? It seems like the vector backbone, after BpiI digestion would already have phosphates on it to mediate ligation, or is the thought that this helps prevent religation of the vector without the inserted oligos?

[Alex Brown]

While the vector remains phosphorylated, the oligos are normally chemically synthesized without. Alkaline phosphatase/CIP aren't needed to remove the vector phosphate because the sticky ends aren't compatible, and therefore are not expected to contribute significantly to circularization of the vector, without a proper insert. Your oligos need to compete with the re-ligation of released insert. You can pay extra for phosphorylated oligos but that will far exceed the cost of PNK after just a few sgRNA. The protocol I listed keeps the annealed oligos in excess of the vector/native insert to help favor the intended ligation, over that of the released insert which remains in the reaction and is already phosphorylated. However, that and the inclusion of the BpiI/BbsI in the one-pot is likely not sufficient to ensure a reasonable efficiency. That being said, you could try it but I would expect to have to screen many colonies before finding anything but empty/original vector from a one-pot protocol like this.

[Sean Collier]

Oligos don't have phosphates st their 5' ends unless if you order them that way.

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