simple method to insert gRNA sequence into pX330 and pX335

[pklein]

Many thanks to Zhang lab for developing tremendous genome editing tools and great web resources. I wanted to suggest a simple cloning method to insert gRNA sequences into pX330 and pX335: Simply combine BbsI digest and ligation in same step (based on "ELAN" method described by Cost and Cozzarelli). This eliminates oligo kinase, vector phosphatase, and gel purification steps. Details follow:

Start with annealed oligo with overhangs as described by Zhang Lab. (Do not phosphorylate oligo).

1.  Digest pX330 or pX335 with BbsI.  (Do not phosphatase treat)

2 µl 10X RE Buffer (e.g. NEB Buffer 2.1)

16 µl H2O

1 µl pX330 (1µg/µl) 

1 µl BbsI

37ºC for 1 hr

 

2.  Add ligation components directly to digest.

Add:  

2.5 µl 10X T4 Ligase Buf

1 µl annealed oligo (10 µM stock; 0.4 µM final concentration)

1.5 µl T4 DNA ligase

37ºC for 1 hr.

 

3.     Transform 2 µl directly from above reaction.

It should also work to add BbsI and ligase components at the same time, but I haven't tried this yet. This method works because the BbsI sites are removed (except in rare cases where the gRNA sequence happens to have a BbsI site). This has worked well for 5 constructs with >90% clones positive for correct insertion.

[agnas]

Thanks for the info. Will let you know if it works!

[Feng Zhang]

Hi,

Thank you so much for sharing the protocol! :)

Feng

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Feng Zhang
Core Member, Broad Institute of MIT and Harvard
Investigator, McGovern Institute for Brain Research
W. M. Keck Career Development Professor in Biomedical Engineering, MIT
zha...@mit.edu

[Warren Wu]

Thank you!

[JP]

What competent cells are you using?

On Wednesday, September 4, 2013 4:28:40 PM UTC-7, pklein wrote:

[JP]

Just wanted to add that I have had success cloning pX330 using the combined technique hinted at below (adding both 10x Buffer 2 and 10x T4 ligase buffer at the same time!) in STBL3 cells. 6/6 colonies with correct insertion. -JP

[pklein]

To JP: We used Z-competent DH5alpha.

Peter

[rp]

What concentration of T4 ligase have you used?

Thanks!

On Wednesday, September 4, 2013 7:28:40 PM UTC-4, pklein wrote:

[pklein]

We use 1.5 ul of NEB T4 DNA ligase (10U/ul x 1.5 ul in 25 ul final = 0.6U/ul final concentration)

[Mohsen Basiri]

Hello all

I used a slightly altered two-step protocol with Fermentas BpiI(BbsI) and T4 DNA ligase and also included a control reaction (without annealed oligos).

Surprisingly, control reaction resulted in many colonies but few colonies were obtained from the main reaction!

My oligo sequence have a RE site; is it worth to analyze some the colonies with this enzyme?.. and basically, what can be the cause of the problem?

Thanks in advance..

[pklein]

I tried adding all components at the beginning and this worked fine in 10/10 colonies/5 different constructs.

[JP]

Using this method your control plates with no insert SHOULD have more colonies than the insert plates. Go for it.

[erikb...@yahoo.com]

I tried this as well, 11 out of 12 screened as positive (I screened 3 each from 4 different constructs), I sequenced 4 of these (1 of each construct), and all were correct.

I love this method. I tried making TALENS before this, and... well, it was a major pain. 

1 anneal step, 1 digest and ligation step, only 3 pieces of DNA (F oligo, R oligo, plasmid) .... 

when I compare this to TALENs... 

36 monomers to pipette, D&L step,  plasmid safe treatment, PCR, gel purifications, 6 hexamars to D&L, transformation... screening many colonies by PCR, then sequencing those colonies by PCR and finding >90% have some point mutation....

I don't think I'll ever try to use TALENs for genome editing again, especially once that double nickase design tool is available.

[Patrick David Hsu]

Hi guys,

We are hard at work on the double nickase design tool. In the meantime, you can easily design guide RNA pairs for double nicking manually. The optimal offset appears to be 0bp-30bp offset between the 5' ends of the sgRNAs. 

Good luck!

Patrick

[cheon...@gmail.com]

That means if I transformed with enzyme cutted plasmid, I have to get more colonies than transformed with annealed plasmid?

 

Thanks in advance...
2013년 10월 1일 화요일 오후 6시 19분 7초 UTC-4, JP 님의 말:

[Sudheendra Rao]

Thanks for sharing.

[rs8...@my.bristol.ac.uk]

Sorry to bring up an old thread, but I was just wondering why you use NEB Buffer 2 for the Bbs1 digest? The 10X FastDigest buffer that comes with Bbs1 has been made so that other downstream enzymes can work in it, such as T4 DNA ligase. Is there some other reason why NEB buffer 2 is chosen over FastDigest buffer?

Thanks! 

[anshika srivastava]

Thanks for sharing the useful information.

Anshika

On Wednesday, September 4, 2013 7:28:40 PM UTC-4, pklein wrote:

[Patrick David Hsu]

Not particularly, the FD 10X buffer works. In fact, it's what I use.

Patrick

[rs8...@my.bristol.ac.uk]

Thanks!

[pklein]

Agreed...no need to use NEB 2.1 specifically. I was using NEB's BbsI at the time and their Buffer 2.1 worked fine. "Fast Digest" is a Thermo brand.
(sorry for the delayed response).
Peter

[Yu Wenhan]

Hi,

May I ask why "Do not phosphorylate oligo"? I can't understand what will happen if i phosphorylate the oligo.

Look forward to your response. Thank you!

John

在 2013年9月4日星期三UTC-4下午7时28分40秒，pklein写道：

[Evan Jones]

Thanks for this Peter, I ran this modified protocol alongside the original Zhang lab protocol for pX458. And both seem to work well with high efficiency, though I'll note, this modified one (as mentioned) saves time and reagents, and actually even appeared to give us higher cloning efficiency. Further sanger sequencing confirmed 8:8 correct cloning inserts!

I appreciate all the help from the Zhang lab and other contributors on the forum, it's great to be a part of such a helpful CRISPR community.

Best,
Evan

[Fatwa Adikusuma]

I am still enjoying the old protocol. We only need to Bbs1 linearize the plasmid once, and we can store and use the linearized plasmid many times to create many different gRNAs. With the modified protocol we need to always digest using Bbs1 every time we make new gRNA which might be costly if we create a lot of different gRNA like I do. The old protocol also saves more time I think. Just my personal opinion. 

cheers,

Fatwa

[m.c.te...@gmail.com]

Since datasheets state that DNA ligases work best at 16 degrees (and that they can loose activity around 37 degrees).. I was wondering whether there's still any use of performing the ligation step at 37 degrees? 

Menno

Op donderdag 5 september 2013 01:28:40 UTC+2 schreef pklein:

[anshika srivastava]

Hi,

I just followed the protocol suggested by peter to clone my sgRNAs in PX462 plasmid. I had 6 different sgRNAs to clone but when I tried to clone out of three sgRNAs I got one successfully cloned in PX462.

So I annealed my oligos (100uM F and 100uM R) as 95C for 2 mins and 25C for 45mins. Then diluted the oligos to 2uM so that in the ligation step my annealed oligos concentration be 0.4uM  

Regarding the PX462 digest and ligation, I am following all the steps as it is. Please suggest.

Thanks

Anshika

On Wednesday, September 4, 2013 7:28:40 PM UTC-4, pklein wrote:

[JP]

Let the reaction go for longer -- 4 hours to overnight if necessary.

[Elitsa]

Big thanks for this method!!!
It worked perfect for me - annealing of the oligos in a big water bath (95C) overnignt, no phosphorylation of the annealed oligo, all in one tube, 2h at 37C, plenty of clones, 10 picked, 10 positive. I used the "normal" BbsI from Thermo with its corresponding buffer, not the fast digest

[方卓清]

 I have a question. Does ligation perform at 37ºC  a good choice? 

在 2013年9月5日星期四UTC+8上午7时28分40秒，pklein写道：

[Kini]

Quick question- I just wanted to make sure you annealed the oligos using the protocol described by the Zhang lab without the addition of PNK so that would be:

1uL of top and bottom oligos
8uL water

into the thermocycler for 37C for 30 min, 95C for 5 min, ramp down to 25C at 5C/min

Thank you so very much for such a helpful protocol! 

On Wednesday, September 4, 2013 7:28:40 PM UTC-4, pklein wrote:

[JP]

Yes that should work fine as long as you use 1 uL of 10x T4 ligase buffer!

[JP]

Ligation works fine at 37C.

[Chris Richie]

I am all for streamlining a protocol, and I am not trying to be a troll, but this method still requires the use of BbsI in every reaction, and that is one more thing to troubleshoot if something goes wrong.  

For labs doing a lot of CRISPR cloning, wouldn't it be better to perform a single "scaled up" BbsI digestion on the sgRNA cloning vector, and dip into that digested backbone for many downstream ligation reactions?

you can still eliminate the kinase, phosphatase, and gel purification steps, although i would heat inactivate the BbsI enzyme before long term storage.

On Wednesday, September 4, 2013 7:28:40 PM UTC-4, pklein wrote:

[Kini]

Hi guys! I am having trouble cloning and was wondering if I could get some advice. My control plate (no insert) has a lot of colonies on it and basically looks like a lawn. The plates with insert in them have less colonies than control but still have what seems like a couple hundred colonies on them. I am scared that maybe I have the wrong plasmid that is not being digested by Bbs1? Has anyone else experienced this and gotten positive colonies?

[JP]

Just dilute it and re-plate. Only way to tell for sure is to do a miniprep and sequence or diagnostic digest.

[Elliott Brea]

Has anyone done this with the lenticrispr protocol ?

[LiHD]

Sorry to bring up this old thread. 

Fatwa, by saying "enjoying the old protocol", did you mean digest big amount of plasimid for multiple experiments first and then do plasmid-annealed sgRNA oligos ligation using T4 liagase? Thanks a lot!

H-D 

[Fatwa Adikusuma]

Yes.

Fatwa

[Anshuman Das]

Hi pklein

I am trying to clone my gRNAs into px334 (which expresses gRNA and tracr RNA separately). My oligos are 20bp each separated by DR and has overhangs as suggested by Zhang lab. I am getting lot of colonies in vector self ligation plate and no positive clones yet. I tried overnight digestion with BbsI and also phosphatase treatment of vector but does not work. Should I follow the same procedure that you described for px335?

Thanks!

Anshuman

[Deba]

Hey pklein,

You mentioned in your combined protocol not to phosphorylate the oligos. So this would be just ramping down the temperature without adding any PNK right?

Thanks,
Deba

[deepika puri]

Hi..

Thanks pklein for the modified protocol...

I am currently just starting with my CRISPR cloning and will try both the original protocol and the modified one...

I just had a question, even using the Zhang lab protocol, why does one need to phosphatase treat the digested vector?

As the site is destroyed after digestion and get purification, there is no chance of vector self ligation right...

Let me know if I'm thinking it all wrong...

so what I'm doing is

oligo: phosphorylate, anneal,

Vector: digest , gel purify,

set up ligation..

[Ran Antes]

Hi everyone.

I have started trying to clone my targets into PX335 using the Zhang protocol and I keep getting empty plates whatever I do.

I am trying to figure what I'm doing wrong but whatever I change nothing helps...

Now I wonder if I can use (in the annealing part) the T4 ligation buffer from thermo, which we have in our lab or the NEB one, which is recommended by the protocol. The thermo one has ~ 10 times more of every ingredient. I have also used the PNK buffer and added 1mM ATP to the reaction but that didnt work...

Thanks for any advise :-)

Ran

[Fatwa Adikusuma]

you were right, no need to dephosporylate if you gel purify or column-based purification. If you purify using precipitation (e.g. Phenol chloroform), there is still chance to self re-ligate.

Fatwa.

[Essen Mark]

Hi,pklein,

I am wondering whether this method can be used for normal clones? I tried but it failed,I am not sure what problem is. It's the problem of this method for normal clone or it' my operation during the clone process? Have you tied this method for normal clones? Thank you!

[xiaoming sun]

Hi JP

Do you mean, when using digestion-ligation simultaneously protocol, 

if you do not put anneal oligo, just plasmid alone, you will have more colonies than experimental one, which has anneal oligo?

Thanks!

On Tuesday, October 1, 2013 6:19:07 PM UTC-4, JP wrote:

Using this method your control plates with no insert SHOULD have more colonies than the insert plates. Go for it.

On Friday, September 20, 2013 12:40:05 AM UTC-7, Mohsen Basiri wrote:

Hello all

I used a slightly altered two-step protocol with Fermentas BpiI(BbsI) and T4 DNA ligase and also included a control reaction (without annealed oligos).

Surprisingly, control reaction resulted in many colonies but few colonies were obtained from the main reaction!

My oligo sequence have a RE site; is it worth to analyze some the colonies with this enzyme?.. and basically, what can be the cause of the problem?

Thanks in advance..

[Alan Aardman]

Our group used this to successfully ligate our gRNA into px330.    We were able to use NEB Quick Ligase instead of the T4 Ligase used by the topic creator.   We just used the recommended concentrations of quick ligase buffer by NEB and the concentrations of oligo proposed here.    It worked well- much better than the traditional gel extraction/ligation protocol you would use.    We were successfully able to produce transgenic mice using px330 synthesized via this method.

Alan

[Abhijit Rath]

Hello pklein,

Thanks for sharing the protocol. However, I am little perplexed about the concn. of oligos. So, is it 1uL of F n R oligos (each at 100uM concn.) being annealed in a 10uL reaction and then you use 1uL of it (without dilution) to get a 0.4uM final concn. (in the 25uL rxn mixture)? If you could clarify that would be great. Thanks