CRISPR-Mediated Base Editing Tools: A Genome Editing Technique to Induce Targeted Base Substitution
Published: April 30, 2023
Abstract
Source: See, J. E., et al. Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors. J. Vis. Exp. (2021).
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 …
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
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Cite This Article
CRISPR-Mediated Base Editing Tools: A Genome Editing Technique to Induce Targeted Base Substitution. J. Vis. Exp. (Pending Publication), e20970, doi: (2023).