By efficiently inducing point mutations using the CRISPR-mediated cytosine bases editor, the functionality of BRCA1 variants, which is important for disease prevention and diagnosis, can be identified. The advantage of this technique is that it directly mutates endogenously expressed BRCA1, which overcomes the limitation of functional evaluation using exogenously expressed BRCA1. Identification of the loss of function mutations of BRCA1 can be used to predict the chance of developing cancers associated with BRCA1 mutations, such as breast, ovarian, prostate, and pancreatic cancers.
It can also be used for finding potential drug targets by searching for essential genes whose viability is reduced through functional depletion. To begin, obtain the BRCA1 genome sequence from GenBank at NCBI and search the 20 base pair target sites with the protospacer adjacent motif sequence, NGGNCCN, around the mutation of interest. The mutation of interest should be located within four to eight nucleotides in the PAM-distal end of the guide RNA target sequences.
Order two complimentary oligonucleotides per guide RNA. For the forward oligonucleotides, add CACCG to the 5'end of the guide sequence and for the reverse oligonucleotides, add AAC to the 5'end and C to the 3'end. After receiving the oligonucleotides, resuspend them in distilled water to a final concentration of 100 micromolar.
Mix the two complimentary oligonucleotides to a final concentration of 10 micromolar with T4 ligation buffer and heat them to 95 degrees Celsius for five minutes then cool them to room temperature for annealing. Digest pRG2 using the restriction enzyme Bsa1 for one hour at 37 degrees Celsius, then run the digested product on a 1%agarose gel and purify the appropriate sized band. Ligate the annealed oligonucleotide duplex to the vector DNA using the purchased DNA ligase according to the manufacturer's protocol and transform them into DH5-alpha competent cells.
Add the transformants to an agar plate containing 100 micrograms per milliliter ampicillin and incubate the plate overnight at 37 degrees Celsius. Purify plasma DNA from several transformants and analyze their guide RNA sequences by Sanger sequencing with primers that prime at the U6 promoter. Seed five times 10 to the fifth 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 purchased transfection reagents according to the manufacturer's protocol. Use one 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 three to four days. Harvest the cell pellets three, 10 and 24 days after transfection to analyze base editing efficiencies.
Extract genomic DNA using the genomic DNA purification kit. Design the first PCR primers to amplify BRCA1 target sites. Although there is no restriction on the size of the first PCR product, a product size of less than one kilobases is recommended to efficiently amplify a specific region.
Design the second PCR primers located inside the first PCR product, taking into consideration the size of the amplicon according to the NGS read length. In order to attach essential sequences for NGS analysis, add additional sequences to the 5'ends of the second PCR primers. Amplify the BRCA1 target sites on the genomic DNA obtained from the three time points using high-fidelity polymerase.
For the first PCR reaction, use 100 nanograms of genomic DNA for amplification over 15 cycles. For the second PCR reaction, use one microliter of the first PCR product for amplification over 20 cycles. Run five microliters of the second PCR product on 2%agarose gel and confirm the size.
Then attach the essential sequences for NGS analysis by amplifying the second PCR product using the primers listed in the text manuscript. Amplify each sample using different primer sets. Use one microliter of the second PCR product for up to 30 cycles of amplification with a high-fidelity polymerase.
Run five microliters of the PCR product on 2%agarose gel to confirm the size and purify the amplicon using a commercial PCR cleanup kit. Mix each sample in equal amounts to create an NGS library. Quantify the NGS library using a spectrophotometer and dilute it to a concentration of one nanomolar using resuspension buffer or 10 millimolar Tris-HCl at pH 8.5.
Prepare 100 microliters of the library diluted to the appropriate loading concentration. As a control combined PhiX with the diluted sample. Load the library onto the cartridge and run NGS according to the manufacturer's protocol.
Obtain over 10, 000 reads per target amplicon for in-depth analysis of base editing efficiency. This protocol was used to perform a functional assessment of endogenous BRCA1 variants generated by CRISPR-based cytosine base editors. CAS-9 and guide RNAs were transfected into HAP1 cell lines to disrupt BRCA1, and mutation frequencies were analyzed.
Mutation frequencies decreased significantly over time in HAP1 cell lines, indicating that BRCA1 is an essential gene for cell viability in these cells. To investigate whether CG to TA substituted variants affect the function of BRCA1, the DNA plasmids and coding guide RNAs that could induce each mutation were transfected to have HAP1-BE3 cell lines. And the substitution frequencies were analyzed.
The relative substitution frequencies of 3598C to T, a pathogenic variant that induces a nonsense mutation, dramatically decreased. Whereas those of 4527C to T, a benign variant that induces a nonsense mutation, remained similar with time. It was found that nucleotide substitution frequencies of these three variants decreased in a time-dependent manner.
Since it is necessary to obtain a high initial mutation frequency in order to clearly recognize the change in cell viability with the passing of the date, it is a priority to establish optimized transfection conditions. This procedure can be applied for verifying other unknown genetic variants such as BRCA VUS. It may provide clues to the pathogenicity of patient-derived unknown variants and their treatment.
