Ultramers vs pcr amplification vs plasmids

[CRISPY-AUS]

Hi all,

once again, back to the basics of CRISPR. I have tried genome editing using CRISPR couple of times in mice.  I do get NHEJ but no where near what is published in the papers. So started to think what could be the reason

1) I am using ultramer oligos for IVT sgRNA. could it be that my sgRNA is inefficient? They look good  on bioanalyzer and nano drop,

2) Cas9 mRNA is good both in quantity and quality.

3) I am doing cytoplasmic injections and transferring 2-cell stage embryos in the recipient mice.  

4) any insight would be great.

Thanks

[Cor]

Hi,

We have been quite successfully with below approach but one thing I learned that (obviously) not all CRISPRs are created equal i.e efficiencies between different CRISPr s can be considerable. We first "validate" the CRISPRs I made by transfecting them to ES cells and check for NHEJ: usually around 2 out of 3 work. We go on with the most promising ones to the zygotes and thusfar we have generated a mouse model from each CRISPr (4 point mutations one LoxP insertion via oligo's and one traditional double strand targeting construct.)

Best,

Cor

Op donderdag 11 december 2014 04:49:58 UTC+1 schreef CRISPY-AUS:

[Prasanna Kallingappa]

Hi Cor,

I am new to this crispr system. I have few questions.

1. Is there any obvious reason/advantage in validating the CRISPRs in ES cells over any cell lines or directly testing by zygote microinjection and validating at the blastocyst level?

2. Since we are also planning to generate a cKO, I would like to know how efficient it is to insert two LoxP sites in both the alleles using the oligos?

3. What is the length of the oliogos that you used as a donor template and how for was the insertion from cut site? 

Thanks in Advance

Prasanna

[Cor]

Op donderdag 11 december 2014 09:58:09 UTC+1 schreef pG:

Hi Cor,

I am new to this crispr system. I have few questions.

1. Is there any obvious reason/advantage in validating the CRISPRs in ES cells over any cell lines or directly testing by zygote microinjection and validating at the blastocyst level?

I guess that a mouse ES cel is as close as we can get to a zygote. We did do validation on injected blastocysts too, and that is the best test off-course.
But zygote injections are more labarious and more importantly much more expensive as ES cell transfections. On top: for ES cells you need plasmid, for zygote injections we use RNA
 

2. Since we are also planning to generate a cKO, I would like to know how efficient it is to insert two LoxP sites in both the alleles using the oligos?

Efficiency is totally locus depended, and as you surly know the change of a cis targeting of both LoxP sites is not that high anyway

 

3. What is the length of the oliogos that you used as a donor template and how for was the insertion from cut site? 

I usually use 50nt homology on both sites, and the desired point mutation and a silent mutation in the spacer region: the last mouse we obtained had the desired point mutation 3 basses down stream of the PAM, and the silent mutation a 11 bases further downstream. In this mouse the silent mutation was NOT incorporated.. (more mice are to be screened though, so it is interesting to see if this phenomena will be observed again)

[pG]

Thanks Cor. That helps.

pG

[CRISPY-AUS]

Hi Cor,

Thanks heaps for your reply. So I am assuming that you are not using ultramers for IVT of sgRNA? As you mentioned that you tested the plasmid in ES cells and then RNA injections in the zygotes. May be I need to switch over to cloning or PCR for IVT. I test the RNA in embryos and genotype them at blastocyst as it is more convenient for me. Surprisingly, in blastocyst I see good NHEJ but same concentration injected and pups screened no positives. 

Are you doing nuclear or cytoplasmic RNA injections? Do you check the quality of your RNA ( bioanalyzer/nanodrop) before injecting? 

No success with point mutation so far. For generating point mutation I have the same template ( 50nt-point mutation-50nt) but so far no success, all though have only screened 12 pups. In one of my template I added HA tag but no success of HR . I am not sure what am I doing wrong? 

[Cor]

Op donderdag 11 december 2014 23:09:24 UTC+1 schreef CRISPY-AUS:

Hi Cor,

Thanks heaps for your reply. So I am assuming that you are not using ultramers for IVT of sgRNA?

..no I use a T7 primer and a reverse primer per the Jeanisch papers and use the subsequent PCR product for the IVT reaction..
 

As you mentioned that you tested the plasmid in ES cells and then RNA injections in the zygotes. May be I need to switch over to cloning or PCR for IVT. I test the RNA in embryos and genotype them at blastocyst as it is more convenient for me. Surprisingly, in blastocyst I see good NHEJ but same concentration injected and pups screened no positives. 

Are you doing nuclear or cytoplasmic RNA injections? Do you check the quality of your RNA ( bioanalyzer/nanodrop) before injecting? 

..my colleague uses cytoplasmatic injections. I do not use the Nanodrop, in my hands these measurements make no sense, instead I run my products on gel next to a RNA ladder with known quantities per band and guestimate my concentration..

No success with point mutation so far. For generating point mutation I have the same template ( 50nt-point mutation-50nt) but so far no success, all though have only screened 12 pups. In one of my template I added HA tag but no success of HR . I am not sure what am I doing wrong? 

..when you see NHEJ there is a change on HR albeit lower, my first point mutated mouse was a single one out of 40 pups tested (about 50% showed NHEJ)..so keep on screening I am afraid

[CRISPY-AUS]

Thanks heaps Cor, really appreciate your feedback. Ya, will get there eventually I guess!!