Overview
This video explains the concept of CRISPR-mediated cytosine base editors for inducing targeted nucleotide substitution.
Protocol
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.
- 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).
- 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.
- 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).
- 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.
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Materials
Name | Company | Catalog Number | Comments |
Lipofectamine 2000 | Thermo Fisher Scientific | 11668027 | Transfection reagent |
Opti-MEM | Gibco | 31985070 | Transfection materials |
FuGENE HD Transfection Reagent | Promega | E2311 | Transfection reagent |
Iscove’s modified Dulbecco’s medium | Gibco | 12440046 | Medium for HAP1 cells |
lentiCas9-Blast | Addgene | 52962 | Plasmids DNA for lentiBE3 cloning |
DNeasy Blood & Tissue Kit | Qiagen | 69504 | Genomic DNA prep. kit |