I am new to CRISPR/Cas9 and I would like to knock-down two different isoforms of the same gene (TFPI) by CRISPR. The isoforms differ in the last exon, which hence becomes an intron of the respective other isoform. I designed CRISPR guides for the last exons. I expect the Cas9 to cut the exon of the first isoform and an intron of the second isoform. What is the expected result when Cas9 cuts in an intron? Will it significantly harm the expression of the gene? If yes, is there a better solution? Thanks for any advice.
I assume you are talking about a conventional CRISPR system with a sgRNA and a functional Cas9, combined with the intentions to introduce a double stranded break in the DNA at the sgRNA specified site.
In general introduction of Indels (the outcome of CRISPR) inside of introns will not affect the expression of the transcript and will have no impact on the functionality of your protein. However, if you direct the CRISPR system in close proximity to any splice related motive (donor, acceptor) you could end up with intron retention or exon skipping.
Just a quick note, introduction of indels in the last exon is not likely to lead to NMD and the introduction of a premature stop codon will only yield a slightly truncated protein, so unless you know that the indels in the last exon is likely to disrupt a functional domain in the protein this will most likely not produce your desired knock-down/knock-out.
In this particular case, I would first create a cell line that with a deletion of the exon-containing intron (using two guides that cut on either sides of the exon to make a deletion). This will (in theory) establish a cell line that will express only one isoform, i.e. the one that uses the last exon. The same principle can be applied to delete only the coding region of the last exon (which should be done in parallel to save time). However, this will most likely be very tricky because if the intron is skipped, it will be difficult to predict what the final product will be if the alternative poly A site is not used. There are other ideas and formats that can be used, but will be more complicated and will require other modifications to the cell line and may not be as efficient.
As Emil pointed out, creating an indel in an intron will not do much, and creating an indel in the final exon of a coding gene will not change expression either.
That being said, after screening for the appropriate clones, it will be important to ensure that there is no funky splicing that is induced (as Emil posted....easy enough to do a quick RT and sequence the resulting mRNAs) and to sequence the deleted areas to make sure that there are no major unintended results. In addition, it would be very useful to start with a clonal parental cell line to do these experiments in order to minimize founder effects.
In theory, this should not affect the general transcription of the gene, only the relevant isoforms that are targeted.
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