if there is only a small sequence difference between the WT and M sequences I think that you have created a heteroduplex of , say, a WT strand annealed to a M band with a bubble os single strand dna in the middle. This bubble causes the strand to migrate through the gel slower than the homoduplex of the same size so gives 3 bands. To test this idea simple add wt and M homoduplex,heat and cool and run at low voltage and the 3rd band should appear. Alternatively isolate the large band ,heat it and let it re anneal and you should generate the 3 bands that you are seeing now.

your picture might support this as the large band looks stronger than the 2 other bands and for a het to form I would expect band ratios of 1:2:1 of N:NM:M

Dear Shuai...

Its clear that the two larger bands were originally one when you do the sequencing, as you used low voltage/more time you gave them a chance to separate! Now, I'm assuming that the sequencing data confirmed your targeted gene! If so, ignore the observation! If not, you have to sequence all the amplified bands!

Best Luck.

if there is only a small sequence difference between the WT and M sequences I think that you have created a heteroduplex of , say, a WT strand annealed to a M band with a bubble os single strand dna in the middle. This bubble causes the strand to migrate through the gel slower than the homoduplex of the same size so gives 3 bands. To test this idea simple add wt and M homoduplex,heat and cool and run at low voltage and the 3rd band should appear. Alternatively isolate the large band ,heat it and let it re anneal and you should generate the 3 bands that you are seeing now.

your picture might support this as the large band looks stronger than the 2 other bands and for a het to form I would expect band ratios of 1:2:1 of N:NM:M

always agree with Paul Rutland

Regards

Hi Shuai Li,

You did not describe the transformation details. I don't know at which stage of the cells you injected the CRISPR-Cas9 components. At 1-cell stage embryo? CRISPR/Cas9 can cause double-stranded breaks (DSB) at the specific locus you tried to knockout. After it causes DSBs, breaks can be fixed through cellular NHEJ repair pathway. The repair results will generate 'INDELs', meaning a few bases will be lost at the site (shorter band when you do PCR), or a few bases will be added (longer band when you do PCR) at the site. If you used a late-stage embryo for injection, you could produce a chimeric mouse parent, some cells are WT, some cells with deleted bases in the specific allele, and some cells with added bases for the allele. Then, you can have 3 bands from the parental line PCR. Chimeric founder lines have been observed before and reported. You should also look into this matter.

**To your question "Can I safety ignore this observation since it does not effect my experiment with the null mouse?" No, I think you should sequence the mysterious band and find out whether extra bases have been added. When you publish the data, the reviewers will ask you the same question anyway.

By the way, did you use embryos for transformation of Cas9 and sgRNA?

@Yau

I like your answer Yau and do appreciate that you have a project on exactly the subject of your answer to this question but if the size change is real due to an insertion it should show at both gel running conditions ( temperature effect?) not just on the 80V runs. I agree the band should be sequenced but think that it is 2 sequences so might need cloning to sequence

Dear Paul,

Thanks for your commet. Yes, I agree with you that he needs to do a cloning for sequencing. However, the copy number of the target gene in the genome can also affact PCR results. So, it is important for Shuai to confirm what this higher band is. Is it a real knockout or is it just a PCR effect?

It is also useful if he can describe how to breed this 'knock-out' parent mouse out. What line did he use to make crosses. I guess he did these crosses: (1) mutant parent mouse x wild = F1 population (individual 1, 2, 3,....), and (2) F1individual 1 x F1individual 2 = pups for PCR experiments

@ Shuai Li,

How many copies of the gene-of-interest in the genome? Only single copy?

Hi Yau: Thank you for the input, sorry I wasn't able to get back on RG sooner. You were correct, I used mutant parent X wild type then F1XF1 just like you said. The animal is not chimeric, I did tissue genotyping with ear, toe and reproductive tissue all showed the same pattern. The cell stage was 1 cell embryo, mouse line wasC57B6/J. I want to thank everyone and let you know that Paul was right, I didn't do exactly what Paul suggested but I ran the PCR with a mixture of WT and homozygous mutant DNA, it turned out to be the same 3 bands. I think in a way it validated the speculation of a heteroduplex. But just to be sure, I will let the Crispr Core group at center for reproductive biology to validate the sequence. I apologize for my lack of knowledge and missing details in the question.

@ Shuai Li,

Since you used embryo at 1-cell stage, not later stage (such as 4-cell stage), then it is unlikely to obatin 'chimeric' founder lines.

And, also, probably you obtained a monoallelic knockout (see attachment***; don't mind the 'Cas9 assay' banding in the figure, they use digestion assay, not PCR). One copy from the homolog chromosomes was mutated and the other copy was still WT. This was deduced from your PCR results of 3 bands using a mixture of WT and homozygous mutant DNA as template.

***Attached figure was obtained from Takara website: (http://www.clontech.com/US/Products/Genome_Editing/CRISPR_Cas9/Technical_Notes/Identification_of_Monoallelic_and_Biallelic_Mutants)

Sometimes, I am thinking how one can find out a CRISPR-mediated knock-out patterns of a specific gene X, if there is a 'pseudogene' of this gene X in the genome. Pseudogenes are genes that have lost their function (ex. truncated, rearrangement,....) which cannot make RNA. CRISPR/Cas9 can target both the 'normal' copy and the 'pseudo' copy as long as the sgRNA-recognized sequence (20-bp) is there. Therefore, I think a Southern analysis should be done first to find out the copy number of the gene-of-interest in the genome before CRISPR/Cas9 is performed.

A question of Pseudogene/CRISPR is on RG: https://www.researchgate.net/post/The_gRNAs_of_cas9_always_off-target_to_pseudogenes_Can_I_use_them

@ Shuai Li,

I am interested in the testing procedure for 'chimerism' of transgenic mouse. You mentioned you tesedt these 3 tissues: ear, toe and reproductive tissue. Is this standard method for test it? When you test their reproductive tissue, will it damage their reproduce ability when you collect the tissue? I guess not, becuase you still want to keep this line for progeny. But I want to know.

Sharing link:

https://books.google.es/books?id=JxC9diKDQMIC&pg=PA383&lpg=PA383&dq=What+are+the+explanations+for+heterozygous+mouse+with+3+PCR+bands?&source=bl&ots=TCqUwNonB3&sig=a6eGLYXqKVaTRUOEjjgRyGo31A4&hl=ca&sa=X&ved=2ahUKEwiQssehk9vZAhUL7RQKHej5Ae4Q6AEwBXoECAIQAQ

Regards!

hi Yau:

I'm not sure if there is a standard procedure for tissues. But for me I use ear, toe, and you only need one side of the reproductive tissue (e.g through a simple ovariectomy). For the Crispr core facility here, they only use 1 cell embryo to minimize chimera animal, but sometimes they still produce chimera, just depends on the crispr/cas9 activity and the techniques used. I cannot tell you in more details because our center for reproductive biology and the crisper core do have a lot patents in generating transgenic animals for research and live stock for big agriculture companies.

Thank you for your answer, Shuai Li.

This was what I really want to know: ["you only need one side of the reproductive tissue (e.g through a simple ovariectomy)"]. So, this tested founder line can still be used for breeding further. Thanks.