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

Samenvatting

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.

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.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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