How to determine where, in the genome, my transgene inserted - (Feb/28/2014 )

I'm making a transgene that's ~5kb that I'm trying to insert into the Rosa26 locus using the CRISPR/Cas9 system.  I blasted a CRISPR guide RNA sequence, that was previously used by Fujii et al, against the mouse genome and although 20 of 20 bases align to the Rosa26 locus, there are several alternative loci that were homologous to the guide RNA at 16-19 bases.  Therefore, insertion into these sites is not impossible.  I want to propagate single cell clones and evaluate where in the genome the transgene inserted.  What's the best way to evaluate this?  I was thinking about making a primer at the end of my transgene and performing sanger sequencing downstream; however, if the gene inserted at numerous sites this could look messy.  Is there a better method?

Well you can use the recirculation trick,

Your transgene comes on a plasmid that can replicate in bacteria. Once the plasmid is integrated into the target locus, you then use a restriction enzyme (that does not cut the plasmid) to digest the integrated genomic DNA. Then use ligase to recircularise plasmid and transform the DNA back into bacteria cell. Re-isolate the plasmid and sequence the genomic DNA that the plasmid captured. You will then know where your transgene has integrated.

Or you try this in theory if you have access to 454 sequencing.

Using a primer of known sequence, amplify from your gene out wards into the genomic region surrounding the transgene. Since there is only one primer, the amplification is linear. Then pass the amplified DNA through beads that have been tethered with a sequence complementary to your primer. The primer sequence and tethered sequence anneal. Then wash off any unbound DNA. Then move the beads to the micro wells in the the 454 sequencing setup and begin sequencing. You will then get the individual sequence of each amplified DNA strand. You will then be able to group the DNA reads together and find out where your transgene has landed.

perneseblue on Sat Mar 1 05:19:43 2014 said:

Well you can use the recirculation trick,

 

Your transgene comes on a plasmid that can replicate in bacteria. Once the plasmid is integrated into the target locus, you then use a restriction enzyme (that does not cut the plasmid) to digest the integrated genomic DNA. Then use ligase to recircularise plasmid and transform the DNA back into bacteria cell. Re-isolate the plasmid and sequence the genomic DNA that the plasmid captured. You will then know where your transgene has integrated.

 

Or you try this in theory if you have access to 454 sequencing.

Using a primer of known sequence, amplify from your gene out wards into the genomic region surrounding the transgene. Since there is only one primer, the amplification is linear. Then pass the amplified DNA through beads that have been tethered with a sequence complementary to your primer. The primer sequence and tethered sequence anneal. Then wash off any unbound DNA. Then move the beads to the micro wells in the the 454 sequencing setup and begin sequencing. You will then get the individual sequence of each amplified DNA strand. You will then be able to group the DNA reads together and find out where your transgene has landed.

Thanks, perneseblue. 

I was not away that people used circularized plasmid for homologous recombination.  I had planned on using linearized DNA, and from what I read this also works.  I would be worried about additional recombination events if coinjecting an entire plasmid along with Cas9.  

I do have access to a 454; however, I was hoping that I could avoid next gen sequencing due to the time, cost, and effort required to perform this. 

Ahrenhase on Sun Mar 2 18:07:15 2014 said:

Thanks, perneseblue. 

 

I was not away that people used circularized plasmid for homologous recombination.  I had planned on using linearized DNA, and from what I read this also works.  I would be worried about additional recombination events if coinjecting an entire plasmid along with Cas9.  

 

I do have access to a 454; however, I was hoping that I could avoid next gen sequencing due to the time, cost, and effort required to perform this. 

 

I need to clarify, the plasmid goes into the cells as a linear molecule. The ends of the linear molecule are homologous to your target. I am not suggesting using circular plasmid in homologous recombination.

If the molecule that you are integrating into the host genome retains the bacteria origin and bacteria selection markers, you can then use the process I described above to cut the plasmid out of the genome by restriction enzymes, recircularise it and put it back into bacteria.

This is the sequence integrated into genome.

5'-homology-Gene of interest-pBR322-AmpR-homology-3'

Then use Restriction enzymes that cuts outside (5'-homology-Gene of interest-pBR322-AmpR-homology-3') on genomic DNA which has integration..

Then use ligase. This will circularise (5'-homology-Gene of interest-pBR322-AmpR-homology-3' +  additional gDNA between homology sequence).

Transform DNA back into bacteria. Only DNA with bacteria origin and selection marker will survive.

Then sequence the gDNA that was captured to know where gene of interest has landed in host genome.

You can also use inverse PCR to isolate the insertion location. Make two outward-directed primers from your transgene insert. Cut genomic DNA with a frequent cutter (4bp recognition site). Religate the cut DNA to circularize fragments, then PCR with the two primers. By sequencing the PCR product, you can identify the genomic insertion site, which will include pieces of both ends of  your transgene (parts adjacent to your primers) and left and right flanking genomic DNA, terminating in the restriction site.