Ethics
This study was approved by the Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University (Approval Reference Number: 2017-49). Written informed consent was obtained from each infertile couple prior to donating immature oocytes for research. Immature oocytes were donated from patients undergoing intracytoplasmic sperm injection (ICSI) from Mar 2015 to June 2017 at the Reproductive Medical Center of the First Affiliated Hospital of Sun Yat-sen University. Written informed consent was obtained from each donor prior to donating immature oocytes for researches. All of the patients followed a protocol using gonadotrophin-releasing hormone agonist and Gonal-F (Gonal-F; Merck Serono, The Netherlands) for ovarian stimulation (Ding et al., 2015). Oocyte retrieval was carried out 34–36 h after the administration of 10,000 IU HCG (Ovidrel; Merck Serono, The Netherlands). Oocytes lacking a polar body were considered immature (germinal vesicle and metaphase I oocytes) after stripping for intracytoplasmic sperm injection (ICSI) on the day of oocyte retrieval. Only the oocytes remaining at the metaphase I stage were used for in-vitro maturation. Written informed consent was also obtained from the β-thalassemia patients to donate blood and skin fibroblast cells for gene editing research in cells and embryos.
Cloning of plasmids
pcDNA3.1(−)-BE3 was synthesized by Guangzhou IGE biotechnology LTD. YEE-BE3 (W90Y/R126E/R132E triple mutant) was from Addgene (#851777). The pcDNA3.1(−)-BE3 was used for expression in human cells and in vitro transcription. pUC19-SpCas9 gRNA expression vector was cloned by amplifying the U6-gRNA fragment from pX330 (Addgene, #42230), and then inserting this fragment into pUC19 vector. Sequences for cloning the gRNA-1, gRNA-2, and gRNA-3 into the pUC19-SpCas9 gRNA expression vector were listed in Table S2. gRNAs was cloned into pDR274 (Addgene, #42250) for in vitro transcription. Sequence for cloning gRNA-1 into pDR274 was listed in Table S3. Target region, spanning HBB −28 sites, was amplified using HBB-FP and HBB-RP primers (Table S4). And then the PCR product was digested with NotI and AscI. This digested PCR product was then cloned into pENTR/D-TOPO vector (Invitrogen), resulting in pENTR/D-TOPO-HBB. −28 A>G mutation was then induced into this vector by quick change PCR using HBB-78-QC-FP and HBB-78-QC-RP primers (Table S4). And then gateway cloning was carried out to clone the −28 (A>G) mutant HBB fragment into pLenti-EF1a-DEST-SFB vector, resulting pLenti-EF1a-DEST-HBB-SFB vector.
Generating HBB −28 (A>G) mutant stable cell line
pLenti-EF1a-DEST-HBB-SFB plasmids were transfected together with psPAX2 (Addgene, #12260) and pMD2.G (Addgene, #12259) into 293T cells to produce lentivirus. 48 h after transfection, the virus was harvested and used to infect 293T cells. 24 h after infection, the infected cells were selected with 1 μg/mL puromycin. After puromycin selection, 3 clones were picked and expanded. The integrated exogenous HBB fragment was amplified and sequenced using FP1 and RP1 (Table S4).
Base editing in HBB −28 (A>G) mutant stable cell line and sequencing
HBB −28 (A>G) mutant stable cell line was transfected with different base editors and gRNAs. Exogenous integrated target sites and 10 potential off-target sites were amplified using primers listed in Table S5. The PCR product was used for TA cloning sequencing or deep sequencing.
Target sites deep sequencing
Ten potential off-target sites were identified by online tool Cas-OFFinder (http://www.rgenome.net/cas-offinder/) to identify potential off-target sites. Sequences surrounding these 10 sites and integrated −28 (A>G) mutation site were PCR amplified and deep sequenced using IlluminaHiseq 2500 PE150 as paired-end 150 reads. The primers for off-target site analysis can be found in Table S3. High-throughput sequencing data was analysed as reported. Briefly, Sample sequencing was done on an IlluminaHiSeq 2000 PE150 as paired-end 150 bp reads. The merged paired-end reads of each library were separated based on barcodes in primers (Table S5) by Python scripts and then submitted to cutadapt (v1.11) for trimming primer sequence. The trimmed reads were aligned to reference sequence by means of BWA with default parameters (v0.7.13). Samtools (v1.3, http://samtools.sourceforge.net) and Picard tools (v2.2.2, http://picard.sourceforge.net) were used to build indices and sort reads. GATK (The Genome Analysis ToolKit, version 3.5) Haplotype Caller and VarScan (v2.4.2, mpileup2snp and mpileup2indel with –min-reads2 10 –min-var-freq 0.01) were used to call variants for all samples and the combined variants of which were then divided into indels and SNVs by SelectVariants. Next, we aligned the reference and repaired sequence to the reads of each barcode by bowtie (version 1.1.2, http://bowtie-bio.sourceforge.net/index.shtmL) with no mismatch. The repair rate was equal to the number of repaired reads divided by the number of reference reads.
Base editing in skin fibroblast cells
1 × 105 skin fibroblast cells was tranfected with 2 μg BE3 expression plasmid and 1 μg gRNA expression plasmid by nucleofection according to the manufacturer’s manual (Lonza, V4XP-2032).
In vitro transcription
BE3 and YEE-BE3 mRNA was transcribed using the mmol/LESSAGEmmol/LACHINE T7 ULTRA kit (Life Technologies) following the manufacturer’s instruction. gRNA-1 transcribed using the MEGAshortscript T7 kit (Life Technologies) following the manufacturer’s instruction. mRNAs and gRNAs were subsequently purified using the MEGAclear kit (Life Technologies) and resuspended in RNase-free water.
In vitro maturation
The in-vitro maturation culture medium consisted of G-IVF medium (Vitrolife Sweden AB, Goteborg, Sweden) supplemented with 10% human serum albumin (HSA) solution (Vitrolife), 25 mmol/L sodium pyruvate (Sigma), 75 IU/L recombinant FSH (Gonal-F; Merck Serono, The Netherlands), and 150 IU/L HCG (Ovidrel; Merck Serono, The Netherlands). Immature oocytes were cultured in a humidified atmosphere of 6% CO2, 5% O2, and 89% N2 at 37°C. The oocyte maturational status was evaluated after 15 h of in-vitro culture. Mature oocytes were identified if they extruded a polar body after 15 h of in-vitro culture and were then used for vitrification. Oocytes remaining immature after 15 h of in-vitro culture were considered incompetent for maturation and were discarded.
Oocyte vitrification and warming
Oocytes in vitro maturation were vitrified and warmed by commercial Kitazato vitrification and warming kit according to the manufacturer’s protocol. Vitrification procedures were performed at room temperature (25–27°C). The oocytes in vitro maturation were transferred from the culture medium into the ES medium (KitazatoBioPharma Co, JP) for 15 min and then VS for 90 s. The oocytes were aspirated and placed on the tip of the Cryotop (KitazatoBioPharma Co, JP) and the Cryotop sheet were plunged into liquid nitrogen immediately. Warming procedures were performed by placing the Cryotop in a warming solution (TS, 1 mol/L sucrose) for 50–60 s at 37°C and moving into a dilution solution (DS, 0.5 mol/L sucrose) for 3 min at room temperature. The oocytes were transferred onto the bottom of WS1 dish with small amount of DS and kept for 5 min in WS1 solution and were then transferred onto the surface of WS2 dish with minimum amount of WS1, then kept for 5 min in WS2 on a plate warmer (37°C).
Enucleation and fusion with donor cells
Thawed oocytes were placed into separate 10 µL manipulation droplets of G-MOPS with 5% HSA and covered with tissue culture oil. After the first polar body of the oocytes reached 12 o’clock, partial zonapellucida dissection (PZD) was performed before enucleation (Ding et al., 2015). Then, they were placed into separate 10 µL manipulation droplets of G-MOPS medium (containing 7.5 µg/mL cytochalasin B, 5% HSA) in a glass-bottom dish at 37°C for 10 min. The spindle was aspirated into the pipette with a minimal amount of cytoplasm and surrounding plasma membrane using Spindle View (Cri Inc.). Enucleated oocytes were rinsed with G-MOPS medium containing 5% HSA and incubated in G-IVF medium with 10% HSA at 37°C in 6% CO2, 5% O2, and 89% N2 for 60 min before fusion. PB1 was aspirated out of ZP by the pipette before enucleated oocyte fused with donor cells. Donor cells were resuspended in a drop containing HVJ-E extract (Cosmo Bio, USA) and were inserted into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were kept in the manipulation medium until cell fusion was confirmed, and then the reconstructed oocytes were transferred into G-IVF medium (10% HSA) and incubated for 1 h before activation.
Artificial activation and embryo culture
The reconstructed oocytes were parthenogenetically activated by incubation in 7.5 mol/L ionomycin (I3909, Sigma, St Louis, MO, USA) for 10 min followed by incubation in 2 mmol/L 6-dimethylamino purine (6-DMAP; d2629, Sigma, St Louis, MO, USA) for 4 h. Activated oocytes with 1 PN were injected G1 gRNA, Cas9 mRNA, and the ssDNAoligo into the cytoplasm 5–6 h after activation. The survived reconstructed embryos were cultured in microdrops of G-1 medium (Vitrolife, VitrolifeSweeden AB Göteborg, Sweeden) at 37°C in a humidified atmosphere of 6% CO2, 5% O2, and 89% N2 for 42 h. Blastomere of reconstructed embryos were individually aspirated out of ZP by the pipette.
Intracytoplasmic injection of BE3 mRNA and gRNA
The mixture of BE3 mRNA (200 ng/μL) and gRNA-1 (100 ng/μL) was injected into reconstructed human embryo about 5 h after activation. And YEE-BE3 mRNA (200 ng/μL) and gRNA-1 (100 ng/μL) was injected into enucleated oocytes after the removal of PB1.
Single embryo PCR amplification and deep sequencing
Single embryo PCR amplification was performed as described before (Zhang et al., 2016). Briefly, each embryo was transferred into a PCR tube containing 1 μL lysis buffer, and then incubated at 65°C for 3 h followed by 95°C for 10 min. The lysis product was then amplified using primers listed in Table S6.
Whole genome amplification by multiplex displacement amplification
Whole genome amplification of the embryos was performed using the PEPLI-g Midi Kit (Qiagen). Briefly, single cell or single blastomere was transferred into PCR tubes containing reconstituted buffer D2 (7 μL), and then incubated at 65°C for 10 min, before the addition of stop solution (3.5 μL) and MDA master mix (40 μL) and incubation at 30°C for 16 h. The DNA preparation was diluted with ddH2O (3:100), and 1 μL of the diluted DNA was used for PCR analysis.