First, you must be able to isolate clones.  This is not straightforward since there generally are no selectable markers in the Cas9 vector system.    In cells, we co-transfect with puro expressing plasmids, grow cells in puro for 48 hours and then plate the survivors by limited dilution in 96 well plates.  Grow up the colonies and use PCR primers to amplify the region around the gRNA recognition site.   You can sequence the PCR products and you should see overlapping sequences, when you hit the breakpoint, if you targeted successfully one allele.   Sometimes you get homozygotes because one repaired allele can be used as a template to repair the second break.  Alternatively, you can clone the PCR products and sequence a few of them to get the sequence of both alleles in your clonal line.  

or try any FLAG-containing constructs, say GFP, then send your cells for sorting, pick up 10-20 clones, check by Western and you can distinguish homo- from hetero-

Thanks for your answers. Actually I want to do a  KO by deleting approximately 50bp. I used an Addgene plasmid with a puro resistance marker so I planned to select clones with puro too. I think I will try Sanger sequencing after Topo TA cloning the PCR products or maybe I can use the Surveyor assay.

Also you can amplify your region of interest around the gRNA recognition site via PCR and test the PCR amplicons with an endonuclease assay, for instance T7E1 or Surveyor Nuclease assay.

Good luck

Jerome

http://www.ncbi.nlm.nih.gov/pubmed/?term=T7E1+surveyor

If you want to delete a bigger region you can also think about a PCR based screen were you have two sets of primers. One pair sourounds your target region. If you have a positive colony you should see a difference on a agarose gel. For the second primerpair, one primer should bind in the deletion region and the other primer upstream or downstream of the target region. Here you would detect negative clones and clones that are heterozyos if they are positive for the first and the second primer pair.

I have had a lot of difficulty with the T7 assay and prefer using a restriction enzyme assay instead. It may be less useful for you since you have already designed everything. What you do is find a restriction enzyme that recognizes the area that will be where the DSB will occur. Once you have PCR the target region you digest the PCR product with the enzyme. Compare with an undigested control. Most likely you will have a lot of heterozygous so there will be a mixture of undigested and digested in the same sample but it is the presence of the undigested band that indicate an indel. If you have the ability to sequence that might be an even better option though.

I have a similar question. Because indel is random, so you may have different indel on each allel, how can you distinguish cells containing one copy knocked out vs two copies by using Sanger sequencing? 

There is a new really convenient and rapid technology available for screening for mutants after CRISPR/Cas9 edited genomic DNA sequences that involves a simple real-time PCR assay using primers that amplify a region 80-100bp that span the the edited locus together with a novel probe technology, Does not involve gel electrophoresis and works when T7 endo and Surveyor methods fail. Using this method you can easily screen large numbers of edited cell populations to determine the frequency of edits. You can detect both HDR and NHEJ gene edits in one simple real-time PCR assay for more info email me at [email protected]

Can we use Colony PCR method to identify which single cell clones has been knockout?

@Pui San Kong, You can use Colony PCR, if you have a double target approach, using two sgRNAs, and cut out a DNA fragment that is large enough to detect the difference between the WT and the deletion allele via colony PCR (e.g. deletion of Exon2). If you use only one sgRNA you might end up with small deletions of 1-30 bps that are not easy to see on a agarose gel, but could be detected via sequencing. 

You can try CE (capillary electrophoresis) based methods without T7EI digestion. For example, IDAA, Fluorescent PCR, CRISPR-STAT. It is based on ABI 3730xl, which is available at many core facilities or companies. It is a fast, sensitive, precise, high-throughput and cost effective approach with single bp resolution. The PCR products do not need to be purified for CE fragment analysis.

The data can be viewed and primary processed by Peak Scanner (free software from Thermo Fisher), and then go to https://www.fragmentanalysis.com for quantitative analysis (more details and references are available from the website).

Hello,

You can first screen your clones by simple Sanger sequencing to distinguish those that have undergone indel events and those that have not. It will first strongly reduce the number of clones to analyze in more details. In my experience with various cell lines sorted by FACS for the presence of Cas9-EGFP, false positive are not very frequent as long as cells are well dissociated. With these conditions, I could confidently directly amplify them and go for WB whenever the antibody allows endogenous protein detection and is raised against relevant epitopes to avoid false negatives for truncated but partially stable products. However, in the case of limiting dilution clonal selection, it is crucial eliminate the excess of unaltered clones before amplification for heavier analyses.

To do this, amplify about 150 bp before and after the sgRNA target site and sequence both sense and antisense. If indels have occured you will see a messy chromatogram corresponding to the superposition of the different alleles you have right from the cut site (between bp 3 and 4 before the PAM motif, see image 1). This however won’t tell you about the exact genotype since indels are quite random.

If you need to know it, NGS is the best approach (Bell et al., 2014 BMC Genomics, PMID: 25409780 or similar strategy). If you have few clones, old school manual NGS with individual PCR product TA-cloning and Sanger sequencing of a library of plasmids for each clone is possible. I don’t have experience with hIPS cells regarding ploidy etc, but for HEK cells clones that could have 2, 3 or 4 copies of the targeted chromosome, it was sometimes difficult to establish clearly the ploidy and genotype since number of NGS reads for each allele was strongly biased in favor of one versus the others. However, this situation can also reflect a more prominent occurrence of a given indel depending on the microhomologies created on the sides of the DSB. To say that 75% reads allele 1, 25% allele 2 can either be a tetraploid clone with 3 times the same indel that is favored because of microhomologies and 1 time the other or a diploid clone with a strong bias in the PCR amplification efficiency in favor of the first.

Anyway, with a combination of sequencing, qPCR/WB and expected phenotypes you’ll be able to say f your model is good.

Good luck

You can use qEva-CRISPR method. Modified clones will present loss of signal proportional to one or two allele modification (compared to unmodified clones).

We have a customized solution to your screening issue called CRISPR-Quest wherein we generate a wild-type blocker XNA oligomer that blocks PCR amplification of unedited templates and only mutant (edited) templates are amplified see attached

Hello

Michael Powell, Can you tell me where is the attached file?

Thanks

Heba

It should be attached now!!

no attachement found Michael Powell

Michael Powell I might be interested to the attachment as well, if it would actually be attached..

Could anyone of you send me Michael Powell's attachment? I am really interested on that.

Hey Daniel Pouly,

Can I know which software or web program you used to visualize and compare control and sgRNA edited sequences in image 1?

Hello,

Sure, it was Sequencher for which the lab had a license.

Thank you Daniel Pouly

Screening for mutations in human induced pluripotent stem cells (hiPSCs) following CRISPR/Cas9 genome editing involves several key steps. These steps are designed to identify both on-target and off-target mutations introduced by the CRISPR/Cas9 system. Here's a professional and detailed approach: ### 1. **Design of CRISPR/Cas9 System**: - Ensure that the guide RNA (gRNA) is designed for high specificity and efficiency. This can be achieved using various online tools that predict gRNA efficiency and off-target effects. ### 2. **Genomic DNA Extraction**: - After the CRISPR/Cas9 editing, extract genomic DNA from the edited hiPSCs. Standard DNA extraction kits or protocols can be used for this purpose. ### 3. **PCR Amplification of Target Sites**: - Amplify the genomic region surrounding the CRISPR/Cas9 target site using PCR. This step is crucial for preparing the DNA for subsequent sequencing. ### 4. **Sanger Sequencing**: - For initial screening, Sanger sequencing of the PCR product can be used. This method allows for the detection of indels (insertions or deletions) at the target site. - The obtained sequences can be compared to the reference sequence to identify mutations. ### 5. **Next-Generation Sequencing (NGS)**: - For more comprehensive analysis, NGS can be employed. This provides a deeper insight into the genome and can detect both on-target and potential off-target mutations with higher sensitivity. - Targeted NGS panels focusing on predicted off-target sites can also be designed. ### 6. **TA Cloning and Sequencing**: - In cases where heterozygous mutations are expected, TA cloning of the PCR products followed by sequencing of individual clones can be useful. This allows for the identification of different alleles. ### 7. **Bioinformatics Analysis**: - Use bioinformatics tools to analyze sequencing data. This involves aligning the sequence data to the reference genome and identifying variants. - For NGS data, specialized software can be used to analyze and filter the data for relevant mutations. ### 8. **Off-Target Analysis**: - Utilize computational tools to predict off-target sites and then validate these sites using targeted PCR and sequencing. ### 9. **Phenotypic Analysis**: - Beyond genetic screening, assess the phenotypic consequences of the edits. This includes cellular morphology, differentiation potential, and functional assays relevant to the hiPSC model. ### 10. **Validation of Clonal Lines**: - After identifying edited clones, it's essential to expand and validate these clones individually. This involves repeating the mutation screening to confirm the genetic alteration and absence of unwanted off-target effects. ### Conclusion Screening for mutations post-CRISPR/Cas9 editing in hiPSCs requires a combination of molecular biology techniques, sequencing technologies, and bioinformatics analysis. The choice of methods depends on the specificity of the gRNA, the nature of the expected edits, and the resources available. It's also crucial to consider the potential for off-target effects and validate the functional integrity of the edited hiPSCs.

l Check out this protocol list; it might provide additional insights for resolving the issue.