Hello I have worked with the same plasmid but did not face any problem. Some tips

Analyse the plasmid and your insert in snap gene.

the bbs1 sites do not self ligate as they are not complementary

you digest the plasmid with bbs1 fast digest from fermentas

gel elute it

anneal oligos as per protocol

no need of PNK and AP treatment

set up ligation reaction as per protocol with T7 dna ligase from neb and do not forget to add ATP.

i have been able to construct two plasmids without any problem.

you can refer to below mentioned paper it is simple, detailed and excellent

CRISPR/Cas9‐based Targeted Genome Editing for the Development of Monogenic Diseases Models with Human Pluripotent Stem Cells

Navin Gupta Koichiro Susa Yoko Yoda Joseph V. Bonventre M. Todd Valerius Ryuji MorizaneFirst published: 26 April 2018 https://doi.org/10.1002/cpsc.50

Hi

I have cloned 6 sgRNAs in px330 and px461 vectors using the same protocol.

In step 1, I perform hybridization and phosphorylation of the complementary sgRNA set using T4 PNK Buffer A and T4 PNK and set the reaction in a thermocycler. Meanwhile i perform digestion of the vector and after digestion i set up ligation reaction using the digested vector reaction (have used 5ul from digestion reaction of 1ug vector and have never gel eluted the digested vector), annealed oligos, NEB T4 Ligase and 1ul of 10mM ATP for 1h at 37 degrees.Following which, i perform transformation and screen the colonies through plasmid digestion using BbsI and AgeI enzymes along with vector, as i compare the digested contruct with digested vector. (The construct should undergo single digestion whereas the vector should undergo double digestion and release about 1kb fragment, so there will be a shift in the fragments of about 1kb ). After this screening, you can proceed further for sequencing.

You can also confirm colonies by pcr using HU6 forward primer and sgrna Rverse.

@Kavita Rawat. The point is that Bbs1 cuts outside it's recogonition sequence, it does not create compatible sticky ends so no chance of recircularization.

Hi Suhail Magray

Exactly, that is why i have used the vector digestion mixture directly for ligation to annealed oligos.

Although, getting colonies in control may be due to improper digestion of the vector. Since, NEB BbsI (if using) needs to be stored at -80, it might not work properly if stored at -20. For the same reason, i switched to fermantas for BbsI enzyme.

Thanks

@Kavita you need not to use PNK or Alkaline phosphotase treatment. These treatments are needed if the vector has compatible sticky ends.

Hi Suhail Magray

I did not get any result initially, when i was not using PNK.

I understand your concern but I have undergone this problem in my initial experiments.

see the job of PNK is to add terminal phosphate to the oligo so that the phosphodiester linkage is formed b/w dephosphorylated vector end which has undergone Alkaline phosphotase treatment. In case you havent used AP what is need of PNK.

Hi everyone,

I'm also dealing with similar problem and I would like to ask about your opinion...

My Vector Backbone (same Plasmid; but cut from different regions for different genes) is varying 3.3kb to 3.4 kb and my inserts sequences are 2.1kb, 2.3kb and 2.3kb.

I tried with;

-1:2, 1:3 ratios,

-used fresh Buffers,

-didnt expose DNA sequences to UV too Long while cutting from gel,

-after isolating DNA sequences from gels, i run them to validate sequences and they were correct too,

-DNA concentrations (Vector Backbone) were changing between 9.5 to 11.5 ng/µl (and I increased Vector DNA amount with insert DNA amount regarding to 1:2 and 1:3 ratios too..)

-At the end of the Ligation, samples were incubated at 16C for overnight +/-18-20hrs

-agar plates were prepared fresh,

I would like to hear your recommendations & ideas..

Cheers,

Z. Ilker

Hi Zeki Ilker Kanbagli

As far i understood your question, I feel you are concerned about the digestion profile of your construct.

If you are determining the length of the DNA fragments based to agarose gel profile and its comparison to DNA ladder, sometimes the problem occurs due to small shift of DNA fragments despite of having correct size.

Or you can check your enzyme for having star activity.

Thanks

Hi Kavita,

I’m sorry, I forgot to specify my problem; I didn’t get colonies at the end...

And my question is instead of performing transfection final constructs into the primary cells with Amaxa, I want to perform aav transfection of my crispr constructs...

(Ps. Another question popped up in my head while brainstorming, maybe I should try sequencing with these DNA sequences that I isolated from gels, what do you think?)

Thanks in advance, waiting for your response!

Cheers,

Z.Ilker

Did you find a protocol that works for BbsI digestion/ligation?

I have the same problem (colonies but with no insert)

Thanks

Ann Sophie

Ann Sophie Moroni do you found an alternative? I am going through the same problem... Tho I realised the digestion/ligation in one step too, being trying separately but it's not working

Paulyna Magaña yes I did!

I used BpiI instead of BbsI, and did not have any problems with that. You can also perform the digestion/ligation step at once.