CRISPR-Mediated Base Editing Tools: A Genome Editing Technique to Induce Targeted Base Substitution

1. Creation of BRCA1 variants using CRISPR-mediated base editing tools

NOTE: If HAP1-BE3 cell lines is not used, BE3-encoding plasmid DNA can be co-transfected with BRCA1-targeting gRNA. Compared to co-transfection of BE3 and gRNA plasmids, transfection of gRNA plasmid to HAP-BE3 cells induce efficient base editing up to 3-fold at target locus in our hands.

1. Seed 5 x 10⁵ HAP1-BE3 cells (or HAP1 cells in case of co-transfection methods) per well in 24-well plates 1 day prior to transfection. At the time of transfection, culture cells to reach an appropriate density (70%–80% confluence).
2. Transfect BRCA1-targeting gRNAs using the purchased transfection reagents (Table of Materials) according to the manufacturer’s protocol. Use 1 µg of BRCA1-targeting gRNAs (with 1 µg of BE3-encoding plasmid DNA in case of co-transfection methods) to induce C:G to T:A conversion at BRCA1 target sites. Incubate the cells at 37 °C and subculture every 3–4 days.
3. Harvest the cell pellets 3, 10, and 24 days after transfection to analyze base editing efficiencies (Samples from 3 days after transfection are analyzed regrading as day 0 samples).
4. Extract genomic DNA using the genomic DNA purification kit (Table of Materials).
   NOTE: We recommend optimizing transfection conditions with variable ratio of reagent to DNA. Optimal condition for HAP1 transfection is 4:1 ration of reagent to DNA in our hands.