Genome editing by saturation of BRCA1 across cell types improves interpretation of variant pathogenicity

This study complies with all relevant ethical regulations. Ethical oversight is maintained for the UK Biobank and BRIDGES. The UK Biobank data was accessed under application 102655 and access to BRIDGES data was approved by the BCAC Data Access Coordination Committee.

Designing and Cloning HDR Libraries

BRCA1 The positions of exons, introns, nucleotides and amino acids are numbered according to RefSeq transcript NM_007294.4.

Each SGE experiment requires a plasmid expressing a gRNA and an HDR plasmid library that provides a repair template for each SNV. Where possible, libraries were designed at Benchling to be compatible with two independent gRNAs. Protospacer adjacent motif (PAM) blocking modifications that did not impact the amino acid sequence were included to prevent recutting of Cas9 after successful HDR editing. For the 5′ UTR and SGE promoter regions, 1 bp and 5 bp deletions at each position were included in addition to the SNVs. Flanking sequences were added to allow PCR amplification.

Oligos were ordered in a pool from Twist Bioscience, amplified by PCR, and purified using AMPure XP (Beckman Coulter). For each SGE region, a homology arm (HA) plasmid was created by PCR amplifying a genomic fragment containing 500–1000 bp of sequence on each side of the SGE region and cloning it into a linearized pUC19 vector using In-Fusion (Takara Bio). HA plasmids were linearized by PCR using primers to create a 15–20 bp overlap with oligopool amplification products, and treated with DpnI (New England Biolabs) to remove the circular plasmid. The amplified oligo libraries were cloned into the linearized HA plasmids using In-Fusion. Competent stellate cells (Takara Bio) were transformed and 1% of each transformation was plated to check efficiency, with the remainder cultured overnight in 150 ml Luria broth containing 100 µg ml⁻¹ carbenicillin. From these cultures, HDR libraries were purified using ZymoPURE Maxipreps (Zymo Research).

The 2018 SGE exon 5 library (ref. ⁷) included PAM-blocking modifications (c.222AG and c.297GA) for two potential gRNAs, but only the 5′ gRNA was used. We designed a new library replacing c.297GA with another synonymous variant disrupting the 5′ target (c.228TC).

The original exon 17 library⁷ did not contain p.R1699Q (c.5096GA) SNV coding because it overlapped with a PAM-blocking modification. We therefore designed a 2 bp variant (c.5096_5097delinsAA) encoding p.R1699Q, which should block redistricting. The exon 17 HA plasmid was modified by in vivo assembly⁶¹ to create an HDR model encoding this variant. This was mixed with the original HDR library at a molar ratio of 1:100 for SGE.

Cloning of gRNA-expressing plasmids

For HAP1 experiments, gRNAs were cloned by ligating annealed oligos into BbsI-digested plasmid pX459.⁶². For HMEC experiments, gRNAs were cloned into pGL3-U6-sgRNA-PGK-blasticidin, derived from pGL3-U6-sgRNA-PGK-puromycin.⁶³ (Addgene, 51133), because the Cas9 HMEC inducible line was resistant to puromycin. Individual gRNAs were cloned by ligating annealed oligos into a BsaI-digested plasmid.

HAP1 cell culture

HAP1 cells with frameshifting LIG4 mutation⁷ were cultured in Iscove’s modified Dulbecco medium (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco). To avoid reversion to diploid, cells were cultured in 2.5 µM 10-desacetyl-baccatin-III (Stratech) and maintained below 80% confluence. For passage, cells were washed in DPBS (Gibco), trypsinized with 0.25% trypsin-ethylenediaminetetraacetic acid (Gibco), neutralized with complete Iscove modified Dulbecco medium, centrifuged at 300°C.g for 5 min and resuspended.

HAP1 SGE protocol

The day before transfection, 18 million cells were seeded per 10 cm plate. Cells were transfected using Xfect (Takara Bio), with the following protocol modifications: 30 μg of gRNA-expressing plasmid, 10 μg of HDR library, and 24 μl of Xfect polymer were combined in 800 μl per transfection. A negative control using the HDR library and a pX459 plasmid expressing gRNA targeting HPRT1a non-essential gene, was carried out for each experiment. Puromycin (Cayman Chemical) was added to the medium at 1 µg ml.⁻¹ on days 1 to 4. At least 6 million cells were passaged on days 5, 8, and 11. On days 5 and 14, pellets of 10 million cells were collected. Negative controls were collected on day 5.

HMEC cell culture

HMEC immortalized by hTERT with doxycycline-inducible Cas9 expression³¹ were cultured in HuMEC medium (Gibco) with 2% FBS and 1% penicillin–streptomycin. For passage, cells were washed in DPBS, trypsinized with trypsin–0.25% ethylenediaminetetraacetic acid, neutralized with defined trypsin inhibitor (Gibco), centrifuged at 180°C.g for 5 min and resuspended in the media.

Generation of a BRCA1
^+/− HMEC line for SGE

To ensure that each diploid cell would only receive one variant, we removed one BRCA1 allele using two gRNAs to cut upstream and downstream of BRCA1. One day before transfection, cells were plated in 12-well plates with 1 μg ml⁻¹ doxycycline (Sigma-Aldrich) to induce Cas9 expression. Cells were transfected with 25 nM of each crRNA:tracrRNA complex (Edit-R, Horizon Discovery) using DharmaFECT 4 (Horizon Discovery) according to the manufacturer’s protocol. Doxycycline was removed from the medium on day 2 and cells were expanded before limiting dilution. To identify lines with a BRCA1 deletion, gDNA was extracted using QuickExtract (Biosearch Technologies). PCR and Sanger sequencing were performed to identify complete monoallelic lineages. BRCA1 deletions and to check for mutations on the remaining alleles (Extended data, Fig. 5a and Supplementary Table 6). A cell line with complete deletion of a BRCA1 allele and a 1 bp upstream deletion (NC_000017.11: g.43125556del) and a 1 bp downstream insertion (NC_000017.11: g.43044294_43044295insG) on the remaining allele were used in the SGE experiments.

HMEC SGE

The day before transfection, 4.2 × 10⁵ Cells were seeded per well in 12-well plates with 1 µg ml⁻¹ doxycycline. Cells were transfected using Xfect using 1.5 µg of HDR library, 1.5 µg of gRNA plasmid, and 1.2 µl of Xfect polymer per well. After 6 h, cells were pooled separately for each replicate. To increase the efficiency of HDR editing, DNA-PK inhibitor NU7441 (Stratech) was added at 300 nM (SGE regions – exons 6, 11 and 10 (3′), intron 11) or 600 nM (SGE regions – promoter, exons 3 and 17). Blasticidin (Gibco) was included in the medium from day 1 to day 5 at 8 µg ml-⁻¹. On day 7, doxycycline and NU7441 were withdrawn. Half of the cells were collected, with the remainder split between untreated and olaparib-treated conditions. Olaparib was included at 300 nM in all HMEC experiments except exons 5 and 10 (5′). For the 5′ exon 12 region, untreated and olaparib-treated experiments were performed using two separate transfections. Cells were passaged every 3 days and collected on days 14 and 21.

Nucleic acid extraction

gDNA and RNA were extracted using the AllPrep DNA/RNA kit (Qiagen), using QIAshredder columns (Qiagen) to homogenize the lysates. gDNA was eluted in 100 μl of nuclease-free water and quantified using the Qubit BR DNA kit (Thermo Fisher Scientific). RNA was extracted from day 5 HAP1 SGE samples and quantified by NanoDrop UV spectrophotometry.

PCR1: amplify gDNA

Each SGE region was PCR amplified from the gDNA using a primer outside of the HAs to prevent amplification of any remaining HDR libraries. Up to 2 µg of gDNA was used per 100 µl reaction, with up to eight reactions per sample and one negative control reaction. KAPA HiFi HotStart ReadyMix (Roche) was used, with MgCl₂ added at 5 mM. SYBR Green (Thermo Fisher Scientific) was added to all reactions at 0.4× to allow for real-time monitoring and termination when completed.

PCR2: adding Nextera adapters

PCR was used to add Nextera adapters onto PCR1 amplicons. Each 25 µl PCR2 reaction used 1 µl of pooled PCR1 product as a template and was run for eight to ten cycles before purification by AMPure XP.

Preparation and amplification of cDNA

For day 5 HAP1 samples, cDNA was prepared from 5 µg of RNA using SuperScript IV first strand synthesis (Invitrogen) using a BRCA1-specific primer. The cDNA was then amplified as described for PCR2 using primers spanning at least one exon junction when possible. For internal regions of exon 10 not spanning exon junctions, RNA was first treated with ezDNAse (Invitrogen).

Indexing and Sequencing

Each sample was doubly indexed by PCR, purified, and quantified using Qubit 1 × dsDNA High Sensitivity (Invitrogen). SGE samples were pooled, assigning 5 × 10 gDNA samples⁶ readings, 3 × 10 cDNA samples⁶ readings, HDR libraries 1 × 10⁶ negative readings and controls 0.5 × 10⁶ bed. Pools were sequenced on an Illumina NextSeq or NovaSeq using 300-cycle paired-end runs.

SGE sequencing data processing

A published pipeline¹² was modified to obtain the number of variants for sample sequencing. Briefly, samples were demultiplexed using bcl2fastq2 (v2.17). SeqPrep was used to merge paired-end reads. Reads were aligned to a reference sequence for each amplicon using EMBOSS “needleall” (v6.6). A custom Python script was used to calculate variant frequencies, annotate editing events, and add CADD (v1.6) annotations. To minimize PCR and sequencing errors, reads required the inclusion of a library variant and at least one PAM blocking modification for calculation of function scores. Variant frequency was calculated by dividing variant reads by the total number of sample reads.

Derive HAP1 function scores

Functional scores were calculated in log form₂ ratio of the day 14 to day 5 frequency of each SNV. Variants with frequency <10⁻⁴ in HDR library or <10⁻⁵ on day 5 were filtered. Variants were also filtered if the functional score difference between replicates was 1.5, unless both scores were <-1.0. Variants resulting in a different amino acid change due to a PAM-blocking modification were excluded. Functional scores were first averaged across replicates and then normalized across regions such that the median nonsense and synonym scores for each coding region matched the overall medians. In cases where variants were initially assigned two scores due to overlapping SGE regions, scores from only one region were used in the overall analysis, although all scores are reported in Supplementary Table 1. Promoter region scores were used for variants overlapping the 5′-UTR region, and exon 12-3′ region scores were used for variants overlapping the exon...