My gene of interest is approximately 2.2 kb long coding for 405 amino acids in length. As this is my first attempt in using CRISPR-Cas9, I am targeting the N-terminus in attempts to delete DNA fragments using Saccharomyces cerevisiae as my model organism. My ideal length to delete is ~25 amino acids.
Is there a limit to the amount of base pairs that can be deleted using CRISPR-Cas9? What would be the recommended amount of base pairs to target for deletion and its efficiency?
You can delete very large fragments in Saccharomyces cerevisiae (larger than 30kb) so your deletion should not be problem and given the efficiency of homologous recombination in Saccharomyces cerevisiae the type of mutation you want to make is quite easy.
I don't have experience using CRISPR in yeast systems, however I have used it quite extensively in mammalian cells and observed deletions of anywhere from 2 - >100 nucleotides (this is via NHEJ DNA repair). One trick I have used to delete larger chunks of DNA, e.g. several exons, is to target Cas9 to two different regions of the gene you're interested in. For example, design sgRNAs for exon 1 and exon 3 and their targeting activity with Cas9 can remove exons 1, 2 & 3 all together at the same time. Best of luck!
CRISPR-Cas9 has been used to delete fragments of up-to 4,5 kilo base pairs. So i think you should be able to use it to delete your desired fragment.
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