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a reliable way to screen F1 mutant fish
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li...@umich.edu
Feb 18, 2019, 11:45:14 AM2/18/19
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Hi, everyone. This is Shuang Li from University of Michigan. I have a problem when i tried to screen F1 mutant fish that was generated by multiple-gRNA injected. I did the PCR and purified the products by doing agarose gel extraction, then send them to sequence, but the the results can not be read. So, I'm just wondering if there are some alternative way to do the screen, except by TA cloning or other cloning way . If anyone knows, please reply me. I appreciate that.
Tamara Stawicki
Feb 18, 2019, 12:36:27 PM2/18/19
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Are you just interested in screening for mutants or do you want to sequence? I have found that I can screen for crispr mutants simply by PCRing ~100bp fragments around the grna target and running out the gel products on a 3% LB (lithium borate) buffer. Usually I get enough separation there to see size changes. Though I have yet to find a good way to sequence F1s without cloning.
On Feb 18, 2019, at 10:13 AM, li...@umich.edu wrote:
Hi, everyone. This is Shuang Li from University of Michigan. I have a problem when i tried to screen F1 mutant fish that was generated by multiple-gRNA injected. I did the PCR and purified the products by doing agarose gel extraction, then send them to sequence, but the the results can not be read. So, I'm just wondering if there are some alternative way to do the screen, except by TA cloning or other cloning way . If anyone knows, please reply me. I appreciate that.
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AlexBarr2
Feb 19, 2019, 1:58:12 PM2/19/19
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El lunes, 18 de febrero de 2019, 17:45:14 (UTC+1), li...@umich.edu escribió:
> Hi, everyone. This is Shuang Li from University of Michigan. I have a problem when i tried to screen F1 mutant fish that was generated by multiple-gRNA injected. I did the PCR and purified the products by doing agarose gel extraction, then send them to sequence, but the the results can not be read. So, I'm just wondering if there are some alternative way to do the screen, except by TA cloning or other cloning way . If anyone knows, please reply me. I appreciate that.
Hello Shuang, what I have done in the past is to PCR a long enough fragment around the target sequence and sequence it from both ends. Then you would have a readable sequence to the point where the deletion (if any) starts and you could estimate very roughly the size of it. Maybe we were lucky, but it worked to find deletions altering the reading frame when we got the homozygous mutants.
> Hi, everyone. This is Shuang Li from University of Michigan. I have a problem when i tried to screen F1 mutant fish that was generated by multiple-gRNA injected. I did the PCR and purified the products by doing agarose gel extraction, then send them to sequence, but the the results can not be read. So, I'm just wondering if there are some alternative way to do the screen, except by TA cloning or other cloning way . If anyone knows, please reply me. I appreciate that.
AKC
Feb 19, 2019, 1:58:32 PM2/19/19
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Synthego's online tool - ICE is pretty good. I've had good success using this tool. The output gives out various predictions (of indels and their frequencies).
One would need sequence traces (.ab1 files) from wildtype (reference) and the G0 (experimental) samples.
https://ice.synthego.com/#/
For general genotyping (without sequencing information), heteroduplex mobility assays (HMAs) using polyacrylamide gel electrophoresis are very instructive. Here are two references that may be useful -
https://www.researchgate.net/publication/303633744_Heteroduplex_Mobility_Assay_HMA_to_analyze_the_mutations
A mixing heteroduplex mobility assay (mHMA) to genotype homozygous mutants with small indels generated by CRISPR-Cas9 nucleases.
Foster SD, Glover SR, Turner AN, Chatti K, Challa AK.
MethodsX. 2018 Nov 27;6:1-5. doi: 10.1016/j.mex.2018.11.017. eCollection 2019.
One would need sequence traces (.ab1 files) from wildtype (reference) and the G0 (experimental) samples.
https://ice.synthego.com/#/
For general genotyping (without sequencing information), heteroduplex mobility assays (HMAs) using polyacrylamide gel electrophoresis are very instructive. Here are two references that may be useful -
https://www.researchgate.net/publication/303633744_Heteroduplex_Mobility_Assay_HMA_to_analyze_the_mutations
A mixing heteroduplex mobility assay (mHMA) to genotype homozygous mutants with small indels generated by CRISPR-Cas9 nucleases.
Foster SD, Glover SR, Turner AN, Chatti K, Challa AK.
MethodsX. 2018 Nov 27;6:1-5. doi: 10.1016/j.mex.2018.11.017. eCollection 2019.
家豪許
Feb 20, 2019, 1:19:06 PM2/20/19
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li...@umich.edu於 2019年2月19日星期二 UTC+8上午12時45分14秒寫道：
Did you
1. performed multiple-gRNA injection to generate multiple genes' knockout and performed PCR amplification with multiple primer pairs?
2.performed multiple-gRNA injection to generate knockout on a single gene and performed PCR amplification with single primer pairs. The sequencing result told you that it is a heterozygous zebrafish, but you could not identify a precise location of the indel?
If the answers is 2., you may identify a mutant with a relative big indel. In our experience, the sequence of the heterozygotes having big indel (more than 18 bp) is relative difficult to be properly sequenced. You can postpone it after you get homozygotes. An easy way to confirm the presents of big indel is amplifying your target gene with a size around 300 bp containing your target sequence. If your candidates showed double bands, while your WT control showed a single band on gel. The one with double bands is the one having big indel. At least, I haven't see exception on our hands. # I personally believe the extra band, the one different from WT, are the hetero-dimer of the PCR product. Though, we never check it.
> Hi, everyone. This is Shuang Li from University of Michigan. I have a problem when i tried to screen F1 mutant fish that was generated by multiple-gRNA injected. I did the PCR and purified the products by doing agarose gel extraction, then send them to sequence, but the the results can not be read. So, I'm just wondering if there are some alternative way to do the screen, except by TA cloning or other cloning way . If anyone knows, please reply me. I appreciate that.
If you do not mind, I will like to confirm your question.
Did you
1. performed multiple-gRNA injection to generate multiple genes' knockout and performed PCR amplification with multiple primer pairs?
2.performed multiple-gRNA injection to generate knockout on a single gene and performed PCR amplification with single primer pairs. The sequencing result told you that it is a heterozygous zebrafish, but you could not identify a precise location of the indel?
If the answers is 2., you may identify a mutant with a relative big indel. In our experience, the sequence of the heterozygotes having big indel (more than 18 bp) is relative difficult to be properly sequenced. You can postpone it after you get homozygotes. An easy way to confirm the presents of big indel is amplifying your target gene with a size around 300 bp containing your target sequence. If your candidates showed double bands, while your WT control showed a single band on gel. The one with double bands is the one having big indel. At least, I haven't see exception on our hands. # I personally believe the extra band, the one different from WT, are the hetero-dimer of the PCR product. Though, we never check it.
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