Allele-Specific Knockdown of Mutant Huntingtin Protein via Editing at Coding Region Single Nucleotide Polymorphism Heterozygosities

Abstract

Huntington's disease (HD) is a devastating, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Inactivation of the mutant allele by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neuronal function. Here, we use a novel HD mouse model to examine allele-specific editing of mutant HTT (mHTT), with a BAC97 transgene expressing mHTT and a YAC18 transgene expressing normal HTT. We achieve allele-specific inactivation of HTT by targeting a protein coding sequence containing a common, heterozygous single nucleotide polymorphism (SNP). The outcome is a marked and allele-selective reduction of mHTT protein in a mouse model of HD. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion/deletion (InDel) mutations and viral vector insertions. Thus, allele-specific targeting of InDel and insertion mutations to heterozygous coding region SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.

Keywords: Huntington's disease; gene editing; single nucleotide polymorphism.

Figure 1.

Strategy for allele-specific targeting of mHTT protein by gene editing. (A) A strategy for allele-specific degradation of mHTT with SNP-specific nucleases. A schematic of the Htt gene depicts 3 of the 67 exons (not drawn to scale). Exon 1 can have a variable number of CAG repeats (indicated in yellow). HD results when a single copy of the gene has a repeat number >36. To specifically alter the disease allele, an allele-specific variable number of trinucleotide (CAG) repeats encoding glutamine (indicated in yellow) nuclease targets an SNP that is heterozygous in an HD individual. In a mouse model of HD, the YAC18 transgene carries a normal version of the human HTT locus with the C allele of SNP RS362331, and the BAC97 transgene carries a mutant human HTT locus and the T allele of SNP RS362331. DNA cleavage and imprecise repair at the mHTT gene result in frameshift mutations that disrupt the coding region of the HD allele. Premature stop codons can reduce expression of the entire HTT protein, possibly through nonsense-mediated decay of the mature mRNA. (B) CRISPR, clustered regularly interspaced short palindromic repeats-Cas9 target sequences specific for two alleles of exon 50 are shown. The heterozygous SNP is shown in blue or red. The PAM sequence is in bold. CAG; CRISPR, clustered regularly interspaced short palindromic repeats; HD, Huntington's disease; HTT, Huntingtin; mHTT, mutant HTT; PAM, protospacer adjacent motif; SNP, single nucleotide polymorphism. Color images are available online.

Figure 2.

Allele-specific targeting of mHTT in BAC97/YAC18 primary neurons. (A) Cartoon depicting the generation and infection of primary cortical neurons from mice heterozygous for Yac18 (wt) and Bac97 (mutant) human transgenes as well as a Cas9 transgene. (B) An allele-specific sgRNA targeting the T allele of Htt Exon 50 (Ex50T) was introduced into BAC97/YAC18/Cas9 mouse primary neurons by lentiviral transduction using the LentiCRISPRcmvGFP vector. An sgRNA targeting the Rosa26 locus was used as a control. Genomic DNA was prepared 7 days after infection. PCR amplicons containing the targeted SNPs were barcoded and analyzed by Illumina sequencing. InDel allele frequencies were determined using the CRISPResso software package. The control sample has a higher frequency of the BAC97 allele than YAC18 due to a higher copy number of the BAC97 transgene. In the treated sample, 40% of reads are induced InDel mutations. n = 3 mice and error bars represent the SEM. p-values <0.05 were considered significant. Exact p-values are reported in Supplementary File 3. (C) The distribution of InDel sizes shows a strong bias toward frameshift mutations (open bars) rather than in frame mutations (filled bars). The insertion of a single base is the dominant allele type. InDel, insertion/deletion; PCR, polymerase chain reaction; SEM, standard error of the mean; sgRNA, single guide RNA; wt, wild type. Color images are available online.

Figure 3.

Allele-specific reduction of mHTT protein in primary neurons from a HD mouse. (A) mHTT and normal HTT protein were assayed after treatment with sgRNAs targeting Exon 50 (Ex50Tg1) or Rosa26 (control). mHTT is expressed from the BAC97 transgene, and normal HTT protein is expressed from the YAC18 transgene. mHTT runs at a higher molecular weight due to the increased size of the polyQ repeat sequence (upper blot). Higher mHTT protein expression is observed because of the higher transgene copy number. Kalirin and beta-tubulin provide total protein concentration controls. The targeted mHTT protein, but not normal HTT protein, is reduced when targeted with sgRNA Ex50Tg1. (B) Quantification of HTT protein levels from digital images of the blot in (A). mHTT and HTT signals are normalized to Kalirin levels. n = 3. Error bars indicate the SEM. Exact p-values are reported in Supplementary File 3. (C) An additional protein gel was overexposed to detect accumulation of truncated protein products corresponding to translational termination after frameshift mutations in exon 50.

Figure 4.

Allele-specific CRISPR-Cas9 targeting of HTT SNPs in the adult striatum of a mouse model of HD. (A) Cartoon depicting the AAV vector used to deliver the Exon50Tg1 and control sgRNAs and the delivery of AAV to mouse brains. The HD model mice have two alleles at the exon50 SNP—the BAC97 transgene with the T allele and the YAC18 transgene with the C allele. The Ex50Tg1 sgRNA targets the T allele of the SNP heterozygosity. The gRNA and a turboRFP reporter gene are delivered by AAV injection into the adult striatum of 8-week-old mice. Cas9 activity is provided as a mouse transgene. (B) Analysis at multiple time points. At 2, 4 and 6 weeks after AAV treatment, the frequency of the BAC97 allele is reduced, and InDel mutations are induced by the Ex50T-programmed nuclease. (C) Analysis of flanking exons. Heterozygous SNPs were examined by Illumina sequencing at two flanking exons in the 4 weeks samples. There is no significant change in the ratio of BAC97 to YAC18 alleles in exons 48 or 57, indicating that cleavage and repair at exon 50 do not induce a high frequency of deletions large enough to remove these SNPs. n = 3 mice and error bars represent the SEM. p-values <0.05 were considered significant. Exact p-values are reported in Supplementary File 3. AAV, adeno-associated virus. Color images are available online.

Figure 5.

Allele-specific reduction of mHTT protein in the mouse adult striatum. (A) Protein sample analysis of HTT and mHTT protein from the striatum of HD model mice with the Cas9, BAC97, and YAC18 transgenes. Each animal was bilaterally injected with AAV expressing the control (Rosa26) or Ex50Tg1 sgRNAs. Protein was isolated from striatal tissue. Vinculin serves as a total protein control. Signal is visualized as a virtual blot format. (B) Quantitative WES signals for mHTT and wt HTT were normalized to vinculin. Each pair of control and Ex50T-treated samples were further adjusted such that the control has a value of 1. At both 4- and 6-weeks after injection with AAV expressing sgRNA Ex50T, mHTT was reduced by >50% while wt HTT was not significantly changed. n = 3 mice and error bars represent the SEM. p-values <0.05 were considered significant. Exact p-values are reported in Supplementary File 3. WES, Simple Western protein analysis.

Figure 6.

Analysis of nonamplified mutations by ddPCR. (A) The relative copy number of human exon 50 was measured using primers (black arrows) and a probe (green arrow) flanking the CRISPR-Cas9 target site. A second set of primers and probe (red arrow) were used to determine the copy number of a reference locus, the mouse RPP30 gene. (B) After DNA cleavage and repair, ddPCR detects chromosomes with either of the original SNP allele sequences or small InDel alleles. (C) ddPCR does not detect chromosomes with mutations that either remove (large deletions) or separate (translations or large insertions) the primer binding sites. (D) ddPCR was used to determine the total copy number of transgenic human HTT exon 50 in the primary neurons using the mouse RPP30 as a reference for the diploid genome. Samples were treated with lentivirus expressing Ex50T gRNA or a control (Rosa26) gRNA. Exon 50 copy number was decreased from an average of 10.2 to 5.7 across three paired neuronal samples. The 44% reduction reflects alleles that can no longer be amplified using the primers for allele sequencing. (E) The fraction of each allele class is estimated by multiplying the fraction of sequenced alleles by the estimated fraction of alleles that could be amplified (0.56) based on the ddPCR results. “non-amp” indicates the estimated fraction of alleles that could not be amplified. (F) ddPCR was used to determine the total copy number of human HTT exon 50 in the 4-week striatal samples treated with AAV expressing the Ex50T or a control (Rosa26) gRNA and in untreated tail DNA from the same animals. There was no significant difference in the exon 50 copy number between tails and striatum treated with the control gRNA. In contrast, the copy number was reduced by an average of 36% in striatum treated with the Ex50T gRNA. This reduction reflects alleles that can no longer be amplified using the primers for allele sequencing. (G) The fraction of each allele class is estimated by multiplying the fraction of sequenced alleles by the estimated fraction of alleles that could be amplified (0.64) based on the ddPCR results. “non-amp” indicates the estimated fraction of alleles that could not be amplified. n = 3 mice and error bars represent the SEM. p-values <0.05 were considered significant. Exact p-values are reported in Supplementary File 3. ddPCR, droplet digital PCR. Color images are available online.