I had similar problems with T7EN1, CelI worked a liitle better for me in the past but recently I have switched to using the TIDE webtool instead as a way to test gRNAs. You sequence the same pcr product you would use for digestion from wt and your treated cells and upload the chromatogram files to the program which use it to calculate indel frequency.

https://tide-calculator.nki.nl/

Hi Yoav,

Thank you for your repply. I know the TIDE workflow. However, as I'm developing a protocol for CRISPR experiments in my species, it would be nice to have a faster and cheaper protocol rather than sequencing. That's why I'm finning tunning the T7EN01 and - soon -  PAGE assays. 

Today I gonna test (1) use less T7 enzyme, 2U and 5U for a 20ul reaction, and (2) performing the assay with the unpurified PCR product.

Best,

Dani.

Dear Daniel,

This answer assumes you are working with diploid cells. The T7 assay could detect also heterozygous SNPs. If this is the case you can send your wild-type PCR for sequencing. If an heterozygous SNP is present, the T7 will cut your annealed PCR product because a bulge is produce where the two variants are annealing incorrectly. Please note that T7 does not make any difference between naturally occurring SNPs and Cas9-induced in-dels. 

You can check on any genome browser if SNPs variants are predicted to be present in your sgRNA target sequence before sending your wt PCR for sequencing. 

Please note also that, should you confirm that a SNP is present in one of your alleles, besides the impossibility to correctly interpret T7 assay results, your sgRNA will target one allele with high efficiency, and the other with a lower one (since the sequence match will not be 100%). In this case the probability of isolating an homozygous clone will be much lower. 

Hope it helps

Cheers

Francesco Aulicino

Dear Francesco,

You are completely right. I sequenced some of my wild type samples and at least 50% have a heterozygous nucleotide site. Yet, this "SNP" is located outside the Cas9 cut site, so it produces a diferent band size after the T7EN01 assay. This means that even that the T7EN01 cuts the wild type sample, the bands migration is different enouth to detect mutants. 

However, I will sequence some of the putative mutant samples and analyze them with TIDE, as suggested by Yoav above, to confirme the T7EN01 assay results. Anyone here have tried using TIDE for mosaic samples? It produce reliable results?

I will also try (probably in the next few weeks) the PAGE-HMA protocol described by Zhu et al., 2014 (An Efficient Genotyping Method for Genome-modified Animals and Human Cells Generated with CRISPR/Cas9 System).

Will be nice to perform all these analysis, so I gonna be able to compare all the main mutant detection strategies and decide the best one for my own data. I will let you guys know about the progress of them.

Best,

Dani.

Dear Daniel,

even if the SNP is not exactly on the sgRNA site it will undermine your result. Consider that if a SNP is present, TN7 will cut 100% of your PCR product. Your sgRNA will induce mutations at a lower frequency than that. If the SNP is close enough to your target site you will keep seeing only the wild-type-like cleavage.

Just out of curiosity how far from the sgRNA site is your SNP? Can you please tell me amplicon size, sgRNA and SNP position starting from the 5' end of your product?

Best

Francesco

Hi Francesco and everyone, 

I'm uploading a figure with a summary of my currently T7EN01 assay experiments. You can find the aswers to your question there. I'm now sanger sequencing some putative mutant individuals in order to analyze them with TIDE.

Keep you guys update ok?!

Bests,

Dani.