Generation of Rhesus Macaque Embryos with Expanded CAG Trinucleotide Repeats in the Huntingtin Gene

Abstract

Huntington's disease (HD) arises from expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene. The resultant misfolded HTT protein accumulates within neuronal cells, negatively impacting their function and survival. Ultimately, HTT accumulation results in cell death, causing the development of HD. A nonhuman primate (NHP) HD model would provide important insight into disease development and the generation of novel therapies due to their genetic and physiological similarity to humans. For this purpose, we tested CRISPR/Cas9 and a single-stranded DNA (ssDNA) containing expanded CAG repeats in introducing an expanded CAG repeat into the HTT gene in rhesus macaque embryos. Analyses were conducted on arrested embryos and trophectoderm (TE) cells biopsied from blastocysts to assess the insertion of the ssDNA into the HTT gene. Genotyping results demonstrated that 15% of the embryos carried an expanded CAG repeat. The integration of an expanded CAG repeat region was successfully identified in five blastocysts, which were cryopreserved for NHP HD animal production. Some off-target events were observed in biopsies from the cryopreserved blastocysts. NHP embryos were successfully produced, which will help to establish an NHP HD model and, ultimately, may serve as a vital tool for better understanding HD's pathology and developing novel treatments.

Keywords: CRISPR; Huntington’s disease; germline editing; rhesus macaque.

Figure 1

Overview of the approach employed for the integration of an expanded CAG repeat region into exon 1 of the rhesus macaque HTT gene. Two sgRNAs were designed to target exon 1 of HTT and the intron between exon 1 and 2 (arrowheads and the associated sequence). A ssDNA was synthesized that served as the donor template DNA containing an expanded CAG repeat, which was injected into rhesus macaque zygotes along with Cas9 protein complexed with the 2 sgRNAs. In cases where ssDNA integration did not occur, the PCR product should have been 508 bp. Embryos with successful ssDNA integration yielded a 710 bp PCR amplicon. Black arrows indicated PCR primers used to amplify the flanking region of HTT gene.

Figure 2

Rhesus macaque blastocysts possessing HDR-mediated expansion of the CAG repeats. (A) A total of 5 TE biopsy samples from blastocysts carried CAG repeats within exon 1 of the HTT gene that differed from wild-type or “unexpanded” CAG repeats. After the amplification of genomic DNA, putative wild-type (508 bp) or HDR (>508 bp) PCR amplicons were detected by means of gel electrophoresis. L, 100 bp ladder; Neg, negative control (no DNA); WT, PCR using genomic DNA of a rhesus macaque from the ONPRC colony. (B) Sanger sequencing results from embryo #1704, which possesses 76 CAG repeats, are representative of the sequencing results. CAG repeats started from bp 151 to bp 378 of the rhesus macaque HTT mRNA, as indicated by the black arrows.

Figure 3

Assessment of genotyping assay accuracy. In order to assess if there is a potential for the ssDNA to lead to the false-positive amplification of CAG repeats during genotyping analysis, the following combinations of ssDNA and genomic DNA were analyzed. Sample 1 contained a mixture of genomic DNA and ssDNA, which was then subjected to WGA. Then, WGA product was used for PCR amplification of HTT gene using primers spanning the HTT gene targeting region. Sample 2 consisted of genomic DNA obtained following WGA and ssDNA that was utilized for HTT gene amplification by PCR. Sample 3 represented the HTT gene amplicon derived from the genomic DNA (WGA product). Neg, a negative PCR control that included no DNA template. L, 100 bp ladder.

Figure 4

Detection of off-target events from two regions. A total of 6 potential off-target regions were PCR amplified using DNA from TE biopsies, with two sites possessing off-target editing events. (A) Examples of editing in a region that is homologous to the sgRNA 2 binding site, referred to as off-target-1. All 5 TE biopsy samples carried an off-target edit on the sgRNA2 off-target-1 region. (B) Examples of editing in a region that is homologous to the sgRNA 2 off-target site 2, referred to as off-target-2. Only one embryo TE biopsy (2382) had a homozygous one-base-pair deletion. The blue highlighted region is the off-target sequence with homology to the sgRNA-2 sequence. The top sequence represents the wild-type unedited sequence obtained from unedited control rhesus macaque DNA. Red arrows denote a mutation.

Figure 4

Detection of off-target events from two regions. A total of 6 potential off-target regions were PCR amplified using DNA from TE biopsies, with two sites possessing off-target editing events. (A) Examples of editing in a region that is homologous to the sgRNA 2 binding site, referred to as off-target-1. All 5 TE biopsy samples carried an off-target edit on the sgRNA2 off-target-1 region. (B) Examples of editing in a region that is homologous to the sgRNA 2 off-target site 2, referred to as off-target-2. Only one embryo TE biopsy (2382) had a homozygous one-base-pair deletion. The blue highlighted region is the off-target sequence with homology to the sgRNA-2 sequence. The top sequence represents the wild-type unedited sequence obtained from unedited control rhesus macaque DNA. Red arrows denote a mutation.