Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Summary

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

Abstract

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Introduction

The breast cancer type 1 susceptibility gene (BRCA1) is a widely known tumor suppressor gene. Because the BRCA1 gene is related to the repair of DNA damage, mutations in this gene would lead to a greater risk of cancer development in an individual¹. Breast, ovarian, prostate, and pancreatic cancers are linked to inherited loss-of-function (LOF) mutations of the BRCA1 gene². Functional assessment and identification of BRCA1 variants may help in preventing and diagnosing the various diseases. To address function of BRCA1 variants, several methods have been developed and broadly used for investigating the pathogenicity of BRCA1 variants such as embryonic stem cell viability assays, fluorescent reporter assays, and therapeutic drug-based sensitivity assays^3,4,5,6. Although these methods have assessed the function of a lot of BRCA1 variants, the methods involving exogenously expressed BRCA1 variants pose limitations in terms of overexpression that might affecting downstream regulation, gene dosage, and protein folding⁷. Furthermore, these assays cannot be harnessed to the posttranscriptional regulation such as mRNA splicing, transcript stability, and effect of untranslated region^8,9.

CRISPR-Cas9 system enables targeted genome editing in living cells and organisms¹⁰. Through a single-guide RNA, Cas9 can induce double-strand breaks (DSBs) in chromosomal DNA at specific genomic loci in order to activate two DNA repair pathways: error-prone nonhomologous end-joining (NHEJ) pathway and error-free homology-directed repair (HDR) pathway¹¹. HDR is a precise repair mechanism; however, DSBs induced by Cas9 nuclease for HDR often results in unwanted insertion and deletion (indel) mutation. Additionally, it needs homologous donor DNA templates for repairing DNA damage and has relatively low efficiency. Recently, Cas9 nickase (nCas9) have been fused with cytidine deaminase domains for targeting C:G to T:A substitutions, without the need for homologous DNA templates and DNA double strand breaks^12,13,14,15. Using the cytosine base editor, we developed a new method for functional analysis of BRCA1 variants¹⁶.

In this study, we used CRISPR-mediated cytosine base editor, BE3¹⁴, which induces efficient C:G to T:A point mutations, for implementing the functional assessment of BRCA1 variants and successfully identified the functions of several BRCA1 variants (Figure 1).

Figure 1: An overview of the workflow for functional assessment. (A) Schematic showing the functional assessment of BRCA1. Because the LOF of BRCA1 affects cell viability, when the BRCA1 mutation is pathogenic, the cells die as the passage number increases. (B) Stages of the functional assessment of BRCA1. Dotted box is optional. It can be replaced by co-transfection of gRNA expressing and BE3 expressing plasmids DNA. Please click here to view a larger version of this figure.

Protocol

NOTE: Method 1 (generation of HAP1-BE3 cell lines) is optional. Instead of constructing a BE3-expressing cell line, BE3-encoding plasmid DNA can be co-transfected with gRNA-encoding plasmid DNA. Other variants of cytosine base editors, such as BE4max, also can be used for highly efficient base editing.

1. Generation of HAP1-BE3 cell lines

1. Construction of plasmid DNA
   1. To construct the lentiBE3-blast plasmid DNA for lentivirus production, amplify the BE3 coding sequences in pCMV-BE3 (Table of Materials) by PCR using high-fidelity polymerase. Design the PCR primer to contain overlapping sequences of the digested vector (from step 1.1.2) for isothermal assembly as follows¹⁷:
      primer-F: 5’-TTTGCCGCCAGAACACAGGACCGGTTCTAGA GCCGCCACCATGAGCTCAGAG-3’,
      primer-R: 5’-CAGAGAGAAGTTTGTTGCGCCGGATCC GACTTTCCTCTTCTTCTTGGG-3’
      NOTE: Nucleotides shown in underlined are overlapped with the digested vector and these sequences will be specific to the destination vector chosen by the user.
   2. Digest lentiCas9-blast (Table of Materials) plasmid DNA with the restriction enzymes, XbaI and BamHI (1 unit per 1 μg) for 1 h at 37 °C. Run the digested product on a 0.8% agarose gel and purify the appropriate-sized bands (8.6 kb) using a commercial gel extraction kit (Table of Materials).
   3. Clone the amplified BE3 PCR product and digested lentiCas9-Blast vector using the isothermal assembly kit (Table of Materials). Use a total of 0.02-0.5 pmol of DNA fragments and a 1:3 ratio of vector:insert. Transform the assembly product into DH5 alpha-competent cells¹⁸. Add the transformants on an agar plate containing ampicillin (100 µg/mL) and incubate the plate overnight at 37 °C.
   4. Pick several colonies and inoculate them in 4 mL of LB (lysogeny broth) medium containing ampicillin (100 μg/mL) and grow the culture in a shaking incubator at 180 rpm.
   5. Purify plasmid DNA using a commercial plasmid DNA purification kit (Table of Materials) according to the manufacturer’s protocols.
   6. To confirm DNA cloning success, analyze the BE3 sequences of each purified plasmid DNA (from step 1.1.5) by Sanger sequencing using standard and BE3-specific primers (Supplementary Table 1) and select the exact cloned lentiBE3-blast construct.
      NOTE: To analyze the results of Sanger sequencing, we recommended several tools such as BLAST (https://blast.ncbi.nlm.nih.gov/) and CLUSTALW (https://www.genome.jp/tools-bin/clustalw).
2. Cell culture and generation of HAP1-BE3 cell lines
   1. Maintain cells in a healthy condition and in an actively dividing state. Culture HAP1 cells in Iscove’s modified Dulbecco’s medium (Table of Materials) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Culture HEK293T/17 cells in Dulbecco’s modified Eagle’s medium (Table of Materials) containing 10% FBS and 1% penicillin/streptomycin.
      NOTE: HAP1 cell is useful for genetic research because of it is nearly haploid cells. However, the cells can spontaneously return to a diploid state in cell culture. Enrichment Hoechst 34580 stained 1n-population using flowcytometry will be helpful for maintenance of haploidy of HAP1 cells¹⁹.
   2. Seed 5 x 10⁶ HEK293T/17 cells on a 100 nm dish 1 day before transfection. On the day of transfection, transfect the plasmid DNA (15 µg of lentiBE3-blast, 9 µg of psPAX2 for viral packaging, and 6 µg of pMD2.G for vial envelope of 2^nd generation lentiviral packaging system) using commercial transfection reagents according to the manufacturer’s protocols (Table of Materials)²⁰. Change the medium at 6 h post-transfection and harvest virus-containing medium at 48 h or 72 h post-transfection. Filter the supernatant using a 0.45 µm filter.
   3. Transduce the lentiviral particles of BE3 into HAP1 cells at a MOI (multiplicity of infection) of 0.1. To adjust for an appropriate MOI, use serially diluted concentrations of virus. Remove the medium and replace it with adjusted viral supernatant and fresh culture medium.
   4. One day after transduction, change the medium to 10 µg/mL of blasticidin (Table of Materials)-containing medium and select the transduced cells for 3 days with blasticidin. After blasticidin selection, select a well with ~10% of surviving cells for the next step.
   5. After blasticidin selection, seed the transduced cells on 96-well plates at a density of 0.5 cells/well in order to isolate single clones (e.g., dilute 50 cells in 20 mL (0.5 cells per 200 µL) culture medium and aliquot 200 µL per well in 96 well plate). Incubate the 96-well plates for 2 weeks and pick the single colonies to confirm the BE3 activity.
   6. Divide the single colonies into two sets, one sets for testing BE3 activity and other for maintenance. Transduce the lentiviral particles of gRNAs into one of the sets to confirm the BE3 activity. Analyze the mutation frequency of each colony using T7 endonuclease I (T7E1, Table of Materials) assay or by performing the targeted deep sequencing technique (step 4)²¹. Select appropriate single clones that are healthy and have a highly active BE3.
      NOTE: To validate exact mutation frequencies, we recommend the targeted deep sequencing than T7E1 assay. HEK2 (5’-GAACACAAAGCATAGACTGCGGG-‘3) is well validated target site for BE3.

2. Design and construction of BRCA1 targeting gRNAs

Figure 2: An example of a gRNA plasmid DNA. (A) To effectively edit the target sequence with BE3, an NGG PAM (CCN PAM) that places the target C (target G) within a five-nucleotide window is required. NGG PAM is shown in red and the base editing window is represented by a gray box. (B) The gRNA sequence for c.8047C>T (H1283Y) base editing is indicated and the target C:G pairs are shown in red while the active window is highlighted by a gray box. For gRNA cloning, the overhang sequences indicated in bold are added at both the 5ʹ ends. Templates for gRNA were generated by annealing the two complementary oligonucleotides. Please click here to view a larger version of this figure.

NOTE: Positive and negative controls of BRCA1 variants are essential. In this study, c.5252G>A (R1751Q) and c.4527C>T (Y1509Y) are used as benign controls. c.191G>A (C64Y), 81-1G>A, and c.3598C>T (Q1200*) are used as pathogenic controls. Target sequences of each gRNA are listed in Supplementary Table 1.

1. Obtain the BRCA1 genome sequence from GenBank at NCBI²².
2. Search the 20-bp target sites with the Protospacer Adjacent Motif (PAM) sequence “NGG” and “CCN” around mutation of interest. The mutation of interest should be located in 4–8 nucleotides in the PAM-distal end of the gRNA target sequences because of the active window of BE3 is 4–8 nucleotides in the PAM-distal end of the gRNA target sequences¹⁴. In the case of c.8047C>T (H1283Y)- and c.5252G>A (R1751Q)-targeting gRNAs, 5ʹ-TCAGGAACATCACCTTAGTG-AGG-3ʹ and 5ʹ-CCA-CCAAGGTCCAAAGCGAGCAA-3ʹ, the sequences in bold are active windows. C to T and G to A conversions occur with NGG and CCN PAM, respectively (Figure 2A).
   NOTE: BE-Designer (http://www.rgenome.net/be-designer/)²³ is useful web-based tools for gRNA design.
3. Order two complementary oligonucleotides per gRNA; for the forward oligonucleotides, add “CACCG” to the 5ʹ end of the guide sequence, and for the reverse oligonucleotides, add “AAAC” to the 5’ end and “C” to the 3’ end. These additional sequences are specific to the destination gRNA expression vector (from step 2.5) used for this protocol, and users should be adjusted for alternative gRNA expression vectors (Figure 2B).
4. Resuspend the oligonucleotide in distilled water at a final concentration of 100 µM. Mix the two complementary oligonucleotides to a final concentration of 10 µM with T4 Ligation buffer (Table of Materials) and heat them at 95 °C for 5 min and cool them at room temperature for annealing.
5. Digest pRG2 using the restriction enzyme, BsaI (1 unit per 1 μg) for 1 h at 37 °C (Table of Materials). Run the digested product on 1% agarose gel and purify the appropriate-sized band (2.5 kb).
   NOTE: Instead of using a classic digestion and ligation method, other cloning method such as Golden Gate cloning can be used.
6. Ligate the annealed oligonucleotide duplex to the vector DNA using the purchased DNA ligase (Table of Materials) according to the manufacturer’s protocol and transform them into DH5 alpha-competent cells (Other E. coli strains which widely used for subcloning also can be used).
7. Add the transformants to an agar plate containing ampicillin (100 μg/mL) and incubate the plate overnight at 37 °C. Purify plasmid DNA from several transformants and analyze their gRNA sequences by Sanger sequencing using primers which prime at U6 promoter (Table of Materials).

3. 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.

4. Sample preparation for Illumina next-generation sequencing (NGS)

Figure 3: Preparation for next-generation sequencing. The 1^st PCR primer was designed to amplify the BRCA1 target site on genomic DNA. The 2^nd PCR primer was designed such that its sequences are located more inside than the 1^st PCR primer sequences. Additional sequences shown as a yellow bar were added at both ends of the 2^nd PCR primer to attach the essential sequences for performing next-generation sequencing. Please click here to view a larger version of this figure.

1. Design the 1^st PCR primers to amplify BRCA1 target sites. Although there is no restriction on the size of the 1^st PCR product, a product size of <1 kb is recommended to efficiently amplify a specific region (Figure 3).
2. Design the 2^nd PCR primers located inside the 1^st PCR product. Consider the size of the amplicon according to the NGS read length (For example, the size of the amplicon product should be smaller than 300 bp for 2 × 150 bp paired-end run to merge each reads). In order to attach essential sequences for NGS analysis, add additional sequences to the 5’ end of the 2^nd PCR primers as follows: for the forward primers, add 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ sequences to the 5’ end, and for the reverse primers, add 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCT-3’ sequences to the 5’ end.
   NOTE: We used nested PCR to reduce the amplicon of non-specific binding by preventing primers from attaching to non-target sequence.
3. Amplify the BRCA1 target sites on the genomic DNA obtained from three time points. Use high-fidelity polymerase according to the manufacturer’s protocol for minimizing PCR errors. For the 1^st PCR reaction, use 100 ng of genomic DNA for amplification over 15 cycles. For the 2^nd PCR reaction, use 1 µL of the 1^st PCR product for amplification over 20 cycles. Run 5 µL of the 2^nd PCR product on 2% agarose gel and confirm the size.
4. In order to attach the essential sequences for NGS analysis, amplify the 2^nd PCR product using the primers listed below. For the PCR reaction, 1 µL of the 2^nd PCR product is used for amplification up to 30 cycles using high-fidelity polymerase.
   1. To perform multiplex sequencing for large numbers of libraries to be pooled and sequenced simultaneously, use forward (D501 – D508) and reverse (D701 – D712) primers, which have unique index sequence (Supplementary Table 1). Amplify each sample using different primers sets to conduct the unique dual indexing strategy.
   2. Run 5 µL of the PCR product on 2% agarose gel to confirm the size, and purify the amplicon using a commercial PCR clean-up kit (Table of Materials). Mix each sample in equal amounts to create an NGS library.
5. Quantify the NGS library at 260 nm wavelength using spectrophotometers and dilute the NGS library to concentration of 1 nM using resuspension buffer or 10 mM Tris-HCl, pH 8.5. Prepare 100 µL of the library diluted to the appropriate loading concentration depending on the library types (For example, prepare 200 pM of the library for Nextera DNA Flex).
   1. As a control, combine phiX with the diluted sample appropriate for the type of kit.
   2. Load the library onto the cartridge and run NGS according to the manufacturer’s protocol. Illumina iSeq 100 or Miseq systems can sequence the amplicons of various length up to 300 bp for single-read or paired end. We recommended over 10,000 reads per target amplicon for in-depth analysis of base editing efficiency.

5. Analysis of base editing efficiency for the functional assessment of BRCA1 variants

1. Analyze the base editing efficiencies using MAUND²⁴. The base editing efficiency is calculated as described below.
   
   If multiple cytosines are present in BE3 active window, only the C to T conversion that is targeted for evaluating the BRCA1 variant is considered. For example, if the sequences of BE3 active window are “C₄A₅T₆C₇T_8,” the possible sequences generated by base editing are “T₄A₅T₆C₇T₈”, “C₄A₅T₆T₇T₈”, and “T₄A₅T₆T₇T₈”. At this time, if position 4 is the targeted position for the desired BRCA1 variant, then only the ““T₄A₅T₆C₇T₈” sequence is considered for calculating base editing efficiency.
   NOTE: We also recommend other web-tools for analysis of base editing activity such as CRISPResso2²⁵, and BE-analyzer²³.
2. Verify the results using positive and negative controls of BRCA1 variants. The base editing efficiencies of the benign control should remain the same, whereas those of the pathogenic control should decrease over time.
3. Calculate the relative base editing efficiency of BRCA1 variants and determine their pathogenicity. The significant differences between Day 0 and Day 21 samples are analyzed appropriate statistical analysis such as t-test.

Results

The experimental approaches described in this protocol enable the functional assessment of endogenous BRCA1 variants generated by CRISPR-based cytosine base editors. To select appropriate cell lines for the functional assessment of BRCA1 variants, researchers should confirm that BRCA1 is essential gene in the targeted cell lines. For example, we first transfected Cas9 and gRNAs into HAP1 cell lines to disrupt BRCA1 and analyzed mutation frequencies by targeted deep sequencing. We found...

Discussion

This protocol describes a simple method for functional assessments of BRCA1 variants using CRISPR-meditated cytosine base editor. The protocol describes methods for the design of gRNAs at target locus and construction of the plasmid DNAs from which they are expressed. Cytosine base editors induce nucleotide conversion in an active window (in case of BE3, nucleotides 4–8 in the PAM-distal end of the gRNA target sequences). The researcher should carefully choose target sequences because all cytosines in acti...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BamHI	NEB	R3136	Restriction enzyme
Blasticidin	Thermo Fisher Scientific	A1113903	Drug for selecting transduced cells
BsaI	NEB	R0535	Restriction enzyme
DNeasy Blood & Tissue Kit	Qiagen	69504	Genomic DNA prep. kit
Dulbecco’s modified Eagle’s medium	Gibco	11965092	Medium for HEK293T/17 cells
Fetal bovine serum	Gibco	16000036	Supplemetal for cell culture
FuGENE HD Transfection Reagent	Promega	E2311	Transfection reagent
Gibson Assembly Master Mix	NEB	E2611L	Gibson assembly kit
Iscove’s modified Dulbecco’s medium	Gibco	12440046	Medium for HAP1 cells
lentiCas9-Blast	Addgene	52962	Plasmids DNA for lentiBE3 cloning
Lipofectamine 2000	Thermo Fisher Scientific	11668027	Transfection reagent
Opti-MEM	Gibco	31985070	Transfection materials
pCMV-BE3	Addgene	73021	Plasmids DNA for lentiBE3 cloning
Penicillin-Streptomycin	Gibco	15140	Supplemetal for cell culture
Phusion High-Fidelity DNA Polymerase	NEB	M0530SQ	High-fidelity polymerase
pMD2.G	Addgene	12259	Plasmids DNA for virus prep.
pRG2	Addgene	104174	gRNA cloning vector
psPAX2	Addgene	12260	Plasmids DNA for virus prep.
QIAprep Spin Miniprep kit	Qiagen	27106	Plasmid DNA prep. Kit
QIAquick Gel extraction Kit	Qiagen	28704	Gel extraction kit
QIAquick PCR Purification Kit	Qiagen	28104	PCR product prep. kit
Quick Ligation Kit	NEB	M2200	Ligase for gRNA cloning
T7 Endonuclease I	NEB	M0302	Materials for T7E1 assay
XbaI	NEB	R0145	Restriction enzyme