Tagging a gene endogenously was previously challenging and had to be done in more crude ways such as using cDNA fusion proteins which can be delivered transiently (AAV) or constitutively (Lenti-virus). However, expression of these constructs is not under control of an endogenous promoter and wont temporally recapitulate the protein. With CRISPR/Cas9, endogenously tagging is now possible due to creating double stranded breaks (DSB) nearly anywhere in the genome in combination with a donor DNA template.
The first decision is what side of the gene you want to tag (start or stop codon) (Figure 1), which might be dependent on:
- sgRNA availability – You want to make sure you have a sgRNA with high cutting efficiency.
- Alternative promoters/UTRs - You want to make sure you tag the main transcript, sometimes not all transcripts will be tagged depending on.
- Viability – Sometimes its safer to tag the 3’ UTR as tagging the start codon can lead to accidental KO via indels on one allele.
Once you have decided which side you will target, the 5’ ATG (C-terminus) or 3’ STOP (N-terminus) of the gene, the next step is positioning of the sgRNA. For the highest chance of your DNA template to integrate into your gene of interest is ensuring the distance between the cut site and START/STOP codon is small and preferably cuts the codon also. Its also important to check the specificity and efficiency of your chosen sgRNA using online tools such as CRISPOR (http://crispor.tefor.net/crispor.py).
When Cas9 creates a DSB, the DNA will attempt to repair itself, leading to a indel and if a donor DNA template with homology arms (HA) that match the DNA flanking the DSB, it will be used as a template to repair and DNA. We can put foreign DNA in between the HA, to insert your desired sequence into the genome. The donor DNA template can be in various formats: plasmid DNA, dsDNA and ssDNA. Each have their own pros and cons.
Most recent gene targeting strategies use ssDNA which integrates via Single stranded template repair (SSTR) as it has the highest efficiency for integration and less chance for random integration, also doesn’t require selection (Figure 2).
When designing a donor template, the sequence you want to insert must be flanked by homology arms and the length of these arms should be between 50-100bp. Its also important to ensure that after SSTR, your sgRNA is not able to cut the genomic DNA again, this can be avoided by using cryptic mutations. There is debate on whether the ssDNA should sense or anti-sense strand in respect to the sgRNA being used, currently there is conflicting evidence on which combination gives the highest SSTR efficiency but both combinations have been shown to work.
The optimal reagents for carrying out SSTR is using Cas9 protein, sgRNA and sgRNA. You can use Cas9 that is expressed from a plasmid but time lag from transcription and translation could result in ssDNA leaving the cell before Cas9 cuts the genomic DNA. These reagents can be introduced via electroporation such as using the NEPA21, Nucleofector or Neon Electroporation system. The settings and reagents will vary depending on cell type and will require optimisation. It’s important to note that SSTR efficiency will correlate with guide activity.
After electroporation you can genotype your pooled or selected clones. When adding a tag, you need to ensure that the whole construct has integrated into the genome.
The first genotyping assay should be an external PCR assay, this uses one primer inside the construct and the one primer outside the right HA, this repeated again designing an assay for the left HA (Figure 3).
Clones positive for both these assays should then be genotyped further. Next, you should check the whole construct in these clones by using two external primers which should give you the whole construct. The PCR product from this assay should be sent for sequencing to check for any errors in repair or indels. Any errors could cause a frameshift and must be checked thoroughly.
Once your clones have passed QC, your clones can be used for experiments! Positive pools can be picked for positive clones.
Asif Nakhuda asif.nakhuda@babraham.ac.uk