Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases

Abstract

Huntington's disease (HD) is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of CAG repeats in the first exon of the huntingtin gene (HTT). The accumulation of polyglutamine-rich huntingtin proteins affects various cellular functions and causes selective degeneration of neurons in the striatum. Therapeutic strategies used to date to silence the expression of mutant HTT include antisense oligonucleotides, RNA interference-based approaches and, recently, genome editing with the CRISPR/Cas9 system. Here, we demonstrate that the CAG repeat tract can be precisely excised from the HTT gene with the use of the paired Cas9 nickase strategy. As a model, we used HD patient-derived fibroblasts with varied numbers of CAG repeats. The repeat excision inactivated the HTT gene and abrogated huntingtin synthesis in a CAG repeat length-independent manner. Because Cas9 nickases are known to be safe and specific, our approach provides an attractive treatment tool for HD that can be extended to other polyQ disorders.

Keywords: CRISPR/Cas9; Huntington's disease; engineered nucleases; genome editing; neurodegenerative diseases; nonsense-mediated decay; repeat expansion.

Figure 1

Pre-screening of HTT_sgRNAs activity in HEK293T cells. (A) Polymorphic CAG repeats and the following CCG repeats are located in exon 1 of the HTT gene. sgRNA1, sgRNA3 and sgRNA4 were designed to target the 5′- and 3′-repeat flanking sequences, respectively. Appropriate PAM sequences are highlighted in green. SgRNA2 uses a non-canonical NAG PAM sequence and targets CAG repeats directly. (B) Illustration depicting the CRISPR/Cas9 expression plasmid used to co-express the SpCas9 protein together with the GFP reporter marker and sgRNA under the U6 promoter. NLS, nuclear localization signal. (C) Analysis of HTT_sgRNA and wt Cas9 activity in HEK293T cells by T7E1 mismatch assays. The signal intensities of the two main bands (marked with an asterisk) was measured. The loss of this signal in relation to the control (100%) was used to calculate the indel frequency (%) in the samples treated with T7E1 (+). The length of the main PCR product is ~ 305 bp. Additional, faster migrating bands in samples non treated with T7E1 enzyme are secondary structure forms of the main product and their contribution is significantly reduced after denaturation of a sample directly before gel electrophoresis (see Figure S1). (D) Sanger sequencing of the HTT gene fragment included the CAG repeat tract. HEK293T cells are heterozygous in this locus with two alleles containing 16 and 17 CAG repeats. After HTT gene editing with CRISPR/Cas9, the sequence trace after the break site comprised a mixture of signals derived from the unmodified and modified DNA.

Figure 2

Excision of the CAG repeats from the HTT gene with paired Cas9 nickases. (A) DNA from HEK293T cells treated with Cas9 nickase and the HTT_sgRNA pairs was PCR amplified and subjected to T7E1 analysis. The length of the unmodified PCR fragment is 305 bp, whereas the edited products are shortened to < 200 bp (marked with an asterisk). (B) A representative Sanger sequencing electropherogram showing the deletion of 107 and 125 bp in HTT_sgRNA1+4- and HTT_sgRNA1+3-treated cells, respectively (marked with an arrow).

Figure 3

Huntingtin inactivation via the Cas9 nickase pair in patient-derived fibroblasts. (A) RT-PCR analysis of the Cas9n/HTT_sgRNA1+4-edited (+) and non-edited (–) HTT gene product in the three human HD cell lines containing 21/44 (GM04208), 17/68 (GM04281) and 21/151 (GM09197) CAG repeats. As a result of repeat excision, shorter PCR products (~545 bp, marked with an asterisk) are present. (B) RT-qPCR analysis of the HTT mRNA levels in the human fibroblast cell lines transfected with the Cas9n/HTT_sgRNA1 and sgRNA4 plasmid pairs. All samples are normalized to human GAPDH, and the results are the mean (± SEM) relative to the cells transfected with the control Cas9n plasmid (one-way ANOVA followed by Bonferroni's post hoc test; the difference was non-significant). (C) Representative western blots showing the HTT protein levels in the control fibroblasts treated with the empty Cas9n plasmids, expressing only Cas9 protein and not sgRNA (C1 and C2) and in the Cas9n/HTT_sgRNA1+4-treated cells (P1 and P2). Plectin was used as the loading control. The lengths of the polyQ tracts in both alleles of the HTT protein in each cell line are marked with an arrow. (D) The quantification of the huntingtin levels in the three human HD cell lines transfected with the Cas9n/HTT_sgRNA1+4 plasmid pair relative to the plectin levels. The results indicate the mean (± SEM) relative to the cells transfected with the control Cas9n plasmid (n = 3; one way ANOVA followed by Bonferroni's post hoc test; ^***p < 0.0001).