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

For CRISPR-mediated genome editing, start with the HAP1-BE3 culture, a haploid cell line with an integrated BE gene encoding a base editor enzyme. To this culture, add lentiviral vectors containing CRISPR-Cas9 plasmid designed for a specific target such as human breast cancer gene, BRCA1.

The lentivirus particle fuses with the host cell membrane, releasing the plasmid, which eventually integrates into the host cell genome to form the CRISPR-Cas9-BE gene segments. These genes generate a BRCA1-targeting gRNA, Cas9 endonuclease, and BE-cytidine deaminase.

The BRCA1-targeting gRNA is an RNA that fuses with Cas9 and directs the complex to the BRCA1 gene locus. Near this gene, locus is a Protospacer Adjacent Motif, or PAM, where BE can stably dock and move upstream to reach its active catalytic region. Here, the BE recognizes a cytidine base and converts it into uridine.

This base substitution mutation triggers Cas9 nuclease to cut the unmodified DNA strand. DNA strand breakage activates repair enzymes that fill in the missing base and convert uracil, a non-DNA base, to thymine. Overall, these processes generate a gene variant.

Now, incubate the cells under physiological conditions and subculture to multiply the gene variant. Extract genomic DNA and sequence it to analyze CRISPR-mediated base editing efficiency.

Seed 5 x 10⁵ HAP1-BE3 cells per well in 24-well plates one day prior to transfection, and culture them to reach 70% to 80% confluence for transfection. Transfect BRCA1-targeting guide RNAs using the purchase transfection reagents, according to the manufacturer's protocol. Use 1 microgram of BRCA1 targeting guide RNAs to induce CG to TA conversion at BRCA1 target sites. Then, incubate the cells at 37 degrees Celsius and subculture every 3 to 4 days. Harvest the cell pellets 3, 10, and 24 days after transfection to analyze base editing efficiencies. Extract genomic DNA using the genomic DNA purification kit.