I am trying to correct a mutation that is a single base-pair insertion, and I need to precisely remove that extra nucleotide to restore the wild-type sequence. I know that larger deletions can be achieved by designing two sgRNAs to generate a double cut, but I am wondering if this is necessary for a single-base deletion. Can a single sgRNA and an ssODN donor template be used to induce an HDR-mediated repair that deletes just one base?
Yes, performing a precise single base-pair deletion using CRISPR/Cas9 is possible using a single sgRNA along with a single-stranded oligodeoxynucleotide (ssODN) donor template to guide homology-directed repair (HDR).
Here is a general approach:
While dual sgRNAs can be used for larger deletions, for a single base change or deletion, a single sgRNA plus ssODN is usually sufficient and more precise.
Keep in mind HDR efficiency can be low depending on the cell type and conditions, so optimizing transfection and using HDR enhancers can improve outcomes.
If you need detailed protocols or references, I can share some useful papers
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