Regulations for research on human gametes and embryos

The regulatory framework surrounding the use of human gametes and embryos for this research was based on the guidelines set by the Oregon Health & Science University (OHSU) Stem Cell Research Oversight Committee (OSCRO). In 2008, OSCRO established policy and procedural guidelines formally defining the use of human embryos and their derivatives at OHSU, informed by the National Academy of Sciences’ Guidelines. These policies and guidelines permitted the procurement of gametes and embryos for research purposes, the creation of human embryos specifically for research, genetic manipulation of human gametes and embryos, creation of human embryonic stem cell lines and molecular analyses. Together, OSCRO and the OHSU Institutional Review Board (IRB) worked concurrently to review and monitor applications for research studies involving human embryos at OHSU.

Human embryo and embryonic stem cell research policies and principles at OHSU were vetted over the course of a decade informed by the NAS guidelines, and subsequently affirmed by new guidelines released in 2015 by the Hinxton Group, the International Society for Stem Cell Research (ISSCR), and 2017 recommendations by the NAS and National Academy of Medicine joint panel on human genome editing.

As part of the review process, OHSU convened additional ad hoc committees to evaluate the scientific merit and ethical justification of the proposed study: the OHSU Innovative Research Advisory Panel (IRAP) and a Scientific Review Committee (SRC). Members of both committees were independent and their names were kept confidential from the research team; OHSU Research Integrity supervised all committee meetings, documentation, and formal recommendations.

Ethical review

While international discussions were in their infancy, the OHSU Innovative Research Advisory Panel (IRAP) Committee was tasked with deliberating ethical considerations related to using gene correction technology in human embryos for basic research at OHSU. The committee was composed of eleven members from internal and external sources: a lay member, a clinical ObGyn physician, three bioethicists, an OHSU Institutional Ethics committee member, three former OSCRO members, a clinical geneticist, and a clinician. Upon completion of the review, the IRAP recommended allowing this research “with significant oversight and continued dialogue, the use of gene correction technologies in human embryos for the purpose of answering basic science questions needed to evaluate germline gene correction prior to the use in human models,” at OHSU.

Study oversight

The established track record of the study team to uphold strict confidentiality and regulatory requirements paved the way for full OHSU IRB study approval in 2016, contingent upon strict continuing oversight which includes: a phased scientific approach requiring evaluation of results on the safety and efficacy of germline gene correction in iPSCs before approving studies on human pre-implantation embryos; external bi-annual monitoring of all regulatory documents regarding human subjects; bi-annual Data Safety Monitoring Committee (DSMC) review; and annual continuing review by the OHSU IRB. The DSMC is required to remain active for the length of the approved IRB protocol and consists of four members: a lay member, an ethicist, a geneticist, and a reproductive endocrinologist. This committee conducts full review of all donations, the subsequent uses of these samples, and participant adverse events. The DSMC provides formal recommendations to the study team and IRB at the completion of each meeting.

Informed consent

The robust regulatory framework set forth by OHSU clearly specified that informed consent could be obtained only if prospective donors were made aware of the sensitive nature of the study. The consent form clearly presented the scientific rationale for the study; stating (in both the Clinical Research Consent Summary and the Purpose section of the consent form) that gene editing tools will be used on eggs, sperm, and/or embryos to evaluate the safety and efficacy of gene correction for heritable diseases. Additionally, consent form language clearly stated that genetic testing would be conducted in addition to creation of preimplantation embryos and embryonic stem cell lines for in vitro analyses and stored for future use. The incidental discovery of genetic information that might be important to the donors’ healthcare is a possible outcome when engaging in this type of research. Informed consent documents provided the donor with the option to receive this information or not. Written informed consent was obtained before all study-related procedures on current, IRB-approved, study-specific consent forms.

Study participants

Healthy gamete donors were recruited locally, via print and web-based advertising. Homozygous and heterozygous adult patients with known heritable MYBPC3 mutations were sought; however, only three adult heterozygous patients were identified by OHSU Knight Cardiovascular Institute physicians and referred to the research team, one of whom agreed to participate in the study.

Controlled ovarian stimulation

Research oocyte donors were evaluated before study inclusion as previously reported; standard IVF protocols and procedures for ovarian stimulation were described previously44. Oocyte donation cycles were managed by OHSU Fertility physicians. Immediately following oocyte retrieval, recovered gametes were transferred to the research laboratory. All study-related procedures took place at the OHSU Center for Embryonic Cell and Gene Therapy. Following oocyte retrieval, cumulus-oocyte complexes (COCs) were treated with hyaluronidase to disaggregate cumulus and granulosa cells. Mature metaphase II (MII) oocytes were placed in Global Medium (LifeGlobal, IVFonline) supplemented with 10% serum substitute supplement (Global 10% medium) at 37 °C in 6% CO 2 and covered with tissue culture oil (Sage IVF, Cooper Surgical).

Compensation

All research donors were compensated for their time, effort, and discomfort associated with the donation process at rates similar to those used for gamete donation for fertility purposes.

Intracytoplasmic sperm injection (ICSI)

MII oocytes were placed into a 50-μl micromanipulation droplet of HTF (modified human tubal fluid) with HEPES 10% medium. The droplet was covered with tissue culture oil. The dish was then mounted on the stage of an inverted microscope (Olympus IX71) equipped with a stage warmer (http://www.tokaihit.com) and Narishige micromanipulators. Oocytes were fertilized by ICSI using frozen and thawed sperm. Fertilization was determined approximately 18 h after ICSI by noting the presence of two pronuclei and second polar body extrusion.

CRISPR–Cas9 injection into zygote or oocytes

For S-phase injections, zygotes were collected 18 h after ICSI and placed in a micromanipulation drop. The CRISPR–Cas9 mixture, containing Cas9 protein (200 ng/μl), sgRNA (100 ng/μl) and ssODN (200 ng/μl), was then injected into the cytoplasm. Injected zygotes were cultured in Global 10% medium at 37 °C in 6% CO 2 , 5% O 2 and 89% N 2 for up to 3 days to the 4–8-cell stage. For M-phase injections, CRISPR–Cas9 was co-injected with sperm during ICSI. A single sperm was first washed in a 4-μl drop of mixture containing Cas9 protein, sgRNA, and ssODN as described above.

Blastomere isolation, whole-genome amplification and Sanger sequencing

Zonae pellucidae from 4–8-cell stage embryos were removed by brief exposure to acidic Tyrode solution (NaCl 8 mg/ml, KCl 0.2 mg/ml, CaCl 2 .2H 2 O 2.4 mg/ml, MgCl 2 .6H 2 O 0.1 mg/ml, glucose 1 mg/ml, PVP 0.04 mg/ml). Zona-free embryos were briefly (30 s) exposed to a trypsin solution (0.15% in EDTA containing Ca- and Mg-free PBS) before manual disaggregation into single blastomeres with a small bore pipette. A total of 830 blastomeres were isolated from 131 embryos, including 19 from control, 54 from zygote-injected and 58 from M-phase-injected groups. Individual blastomeres were transferred into 0.2-ml PCR tubes containing 4 μl PBS and placed into a freezer at −80° C until further use. Whole-genome amplification was performed using a REPLI-g Single Cell Kit (Qiagen). Successful amplification was evaluated by PCR for MYBPC3 and Sanger sequencing. Briefly, amplified DNA was diluted 1/100 and the on-target region for MYBPC3 was amplified using a PCR Platinum SuperMix High Fidelity Kit (Life Technologies) with primer set: F 5′-CCCCCACCCAGGTACATCTT-3′ and R 5′-CTAGTGCACAGTGCATAGTG-3′. PCR products of 534 base pairs (bp) were purified, Sanger sequenced and analysed by Sequencher v5.0 (GeneCodes). Of 830 blastomeres, 730 (88%) resulted in successful libraries and produced PCR products for MYBPC3 while the remaining 100 blastomeres (12%) failed to generate PCR products and were excluded from the study.

iPSC derivation and transfection with CRISPR–Cas9

Patient iPSCs were derived from skin fibroblasts with a CytoTune-iPS Reprogramming Kit (Life Technologies), according to the manufacturer’s protocol. Cell lines were cultured in mTeSR1 medium (STEMCELL technology) at 37 °C in a humidified atmosphere containing 5% CO 2 . To test CRISPR–Cas9, 2 × 105 iPSCs were dissociated into single cells (using Accutase from STEMCELL technology, or TrypLe from Invitrogen). For the CRISPR–Cas9-1 construct (in the J.-S.K. laboratory), Cas9 expression plasmid (p3 s-Cas9HC, 2.4 μg), sgRNA expression plasmid (pU6-sgRNA, 1.6 μg) and ssODN-1 (100 pmol, IDT) were transfected into iPSCs using an Amaxa P3 Primary Cell 4D-Nucleofector Kit (Program CB-150) according to the manufacturer’s protocol. Three days after transfection, ~5,000 cells were plated onto a Matrigel-coated culture dish and cultured for clonal propagation and individual clone selection. For the CRISPR–Cas9-2 construct (in the J.C.I.B. laboratory), Cas9 expression plasmid (pCAG-1BPNLS-Cas9-1BPNLS, 15ug), sgRNA expression plasmids (pCAGmCherry-MYBPC3gRNA, 15ug) and 30 μg ssODN-2 (IDT) were co-transfected by electroporation using the BioRad Gene Pulser II (a single 320-V, 200-μF pulse at room temperature) with a 0.4-cm gap cuvette. Cells were plated at high density on 6-well plates coated with Matrigel. Two to three days after electroporation, iPSCs were harvested and subjected to clonal selection. All cell lines were negative for mycoplasma contamination. For direct comparisons of CRISPR–Cas9-1 and CRISPR–Cas9-2, Cas9 RNP complexes, composed of the recombinant Cas9 protein (15 μg) and sgRNA (20 μg), were co-transfected with ssODN-1 (50–200 pmol, IDT) into iPSCs (2 × 105 cells) via electroporation as described above. Three days after transfection, indel and HDR efficiencies were analysed by targeted deep sequencing.

Recombinant Cas9 protein and in vitro transcription of sgRNA

Recombinant Cas9 protein was purchased from ToolGen, Inc. The sgRNA was synthesized by in vitro transcription using T7 polymerase (New England Biolabs) as described previously45. In brief, sgRNA templates were generated by annealing and extension of two oligonucleotides (Extended Data Table 1). Then, in vitro transcription was performed by incubating sgRNA templates with T7 RNA polymerase supplemented with NTPs (Jena Bioscience) and RNase inhibitor (New England Biolabs) overnight at 37 °C. In vitro transcribed RNA was then treated with DNase I (New England Biolabs) for 30 min at 37 °C, and purified using MinElute Cleanup kit (Qiagen).

Targeted deep sequencing, genomic DNA cleavage, WGS and Digenome sequencing

To analyse HDR and NHEJ frequencies, on-target and off-target regions were amplified using Phusion polymerase (New England Biolabs). PCR amplicons were subjected to paired-end sequencing using Illumina Miniseq. Cas-analyzer was used for analysing indel and HDR frequencies46,47. The primers used for amplification are listed in Extended Data Table 5. Genomic DNA was isolated from patient iPSCs using a DNeasy Tissue Kit (Qiagen). Digenome-seq was performed as described25,26. In brief, 20 μg genomic DNA was cleaved by incubating recombinant Cas9 protein (16.7 μg) and in vitro transcribed sgRNA (12.5 μg) in 1× NEB buffer 3.1(100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg/ml BSA, pH 7.9) at 37 °C for 3 h. Cas9- and sgRNA-treated genomic DNA was treated with 50 μg/ml RNase A (Sigma Aldrich) at 37 °C for 30 min, and purified with a DNeasy Tissue Kit (Qiagen). WGS and Digenome sequencing were performed as described previously25,26. In brief, 1 μg genomic DNA was fragmented and ligated with adaptors using TruSeq DNA libraries. DNA libraries were subjected to WGS using an Illumina HiSeq X Ten Sequencer at Macrogen (30× to 40×). The sequence file was aligned to the human reference genome hg19 from UCSC with the following mapping program and parameters using Isaac aligner48: base quality cutoff, 15; keep duplicate reads, yes; variable read length support, yes; realign gaps, no; and adaptor clipping, yes (adaptor: AGATCGGAAGAGC*, *GCTCTTCCGATCT). In vitro DNA cleavage sites were identified computationally using a DNA cleavage scoring system described previously26. Indel frequencies of 23 genomic loci with DNA cleavage score above the 0.1 cutoff value were individually examined in individual blastomeres by targeted deep sequencing. Primers for detecting indel frequencies of Digenome-seq captured sites are listed in Extended Data Table 5.

Analysis of off-target effects in CRISPR–Cas9-injected human embryos by WGS

WGS was performed using an Illumina HiSeq X Ten sequencer with a sequencing depth of 30× to 40× (Macrogen, South Korea). Sequences from each blastomere were processed to obtain total variants (lane 1 in Extended Data Table 6) using the Isaac variant calling program48. Annotated variants, including dbSNPs and all novel SNPs (substitution changes), were filtered out, and novel indel sites were identified (lane 2 in Extended Data Table 6). Cas-OFFinder46 was used to extract potential off-target sequences that differed from the on-target sequence by up to 7-nucleotide mismatches or up to 5-nucleotide mismatches with a DNA bulge of up to 2 nucleotides. Indel sites found in each blastomere were compared to homologous sites identified by Cas-OFFinder and potential off-target sites were identified (lanes 3 and 6 in Extended Data Table 6). Then, we excluded potential off-target sites, which were found in intact control embryos (C2.3 and C10.2, lanes 4 and 7 in Extended Data Table 6). Finally, we determined whether each of these potential off-target sites was caused by CRISPR-Cas9 by inspecting sequences with Integrative Genomics Viewer27 (IGV, lanes 5 and 8 in Extended Data Table 6).

Whole-exome sequencing and data analyses

Whole-exome sequencing (WES) was performed using genomic DNAs isolated from peripheral blood of the sperm donor and two egg donors (egg donor1 and egg donor2) and ES cells derived from individual human embryos (ES-WT1, ES-Mut1 and ES-C1 were from egg donor1; ES-WT2 and ES-WT3 were from egg donor2). ES-WT1, ES-WT2 and ES-WT3 were from treated wild-type embryos. ES-C1 was from an untreated wild-type embryo. ES-Mut1 was from a treated heterozygous mutant embryo. Sequencing libraries were prepared according to the instructions for Illumina library preparation. Exome capture was done using an Agilent V5 chip. Sequencing was done on an Illumina Hiseq 4000 platform with paired-end 101 (PE101) strategy at a depth of 100×. All sequencing data were first processed by filtering adaptor sequences and removing low quality reads or reads with a high percentage of N bases using SOAPnuke (1.5.2) software (http://soap.genomics.org.cn/) developed by BGI, and clean reads were generated for each library. Clean data were paired-end aligned using the Burrows-Wheeler Aligner (BWA) program version 0.7.12 to the human genome assembly hg19. Duplicate reads in alignment BAM files were identified using MarkDuplicates in Picard (1.54). The alignment results were processed by RealignerTargetCreator, IndelRealigner and BaseRecalibrator modules in GATK (3.3.0). Variant detection was performed using HaplotypeCaller tool in GATK. SNV and indel information was extracted and filtered by VQSR in GATK and annotated by AnnoDB (v3).

The guide sequence (GGGTGGAGTTTGTGAAGTAT) was aligned to the human genome assembly hg19 to identify potential off-target sites by full sensitive aligner Batmis (V3.00), allowing a maximum of five mismatches globally and a maximum of two mismatches in the core region (12 bp adjacent to the PAM site). Inherited variants from parents and all novel SNPs (substitution changes) were filtered out, and novel indels located within the off-target site plus flanking 20-bp region were defined as off-target variants.

Statistical analyses

Student’s t-test was used for the comparisons in Fig. 4a. One-tailed Fisher’s test was used for the comparisons in Fig. 2f, Fig. 3c, and Extended Data Fig. 1e. One-way ANOVA with Bonferroni test was used for the comparisons in Extended Data Table 2. A P value less than 0.05 was considered significant. No statistical methods were used to predetermine sample size. The experiments were randomized and the investigators were blinded to allocation during experiments and outcome assessment whenever possible.

Data availability

The sequencing data sets, including WGS, WES and Digenome-seq, generated during the current study are not being made publicly available owing to concerns that the data could reveal the research participant’s genetic identity, and revealing the identity would be against the participant’s wishes and consent. However, the data will be made available to researchers from the corresponding author on reasonable request, dependent upon OHSU IRB approval.