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A Breakthrough of gene editing assisted by Annoroad ——the creation of the world’s first pig model for Huntington’s disease


A team of scientists from Jinan University(JNU) has created the world’s first pig model with Huntington’s disease (HD) by using CRISPR/Cas9 and somatic nuclear transfer technology. This study was published in Cell on March 29th. Annoroad conducted whole genome sequencing of the pig genomic DNA and prepared the DNA library. It is demonstrated for the first time that overt and typical neurodegeneration seen in human brains can be endogenously expressed in large mammals, which offers great hope to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.

Figure 1. Scientists with the world’s first pig models with Huntington’s disease.

HD together with Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) are all age-dependent neurodegenerative diseases, which are caused by the accumulation of misfolded proteins in the brain. HD results from the mutation of a single gene HTT, the feature of that makes it an ideal model for the exploration of multiple neurodegenerative diseases. Although mouse models have been established and widely used in the study of HD, there are still considerable differences between human and rodent brains, leading to an absence of overt and striking hallmark of HD. Compared with other small mammals, pigs are genetically, anatomically and physiologically closer to humans.

The research team led by Professor Xiaojiang Li designed two guide RNAs (gRNAs) to target the pig HTT intron after exon 1 to promote DNA breaks and replaces pig HTT exon 1 with the human exon 1 (containing a 150-CAGs repeat) via homologous recombination. The donor vector carries human exon 1 HTT with 150-CAGs repeat flanked by two homologous pig HTT sequences (1Kb for each left and right arm). Afterward, the gRNAs, Cas9 and the donor vector were used to transfect fetal pig fibroblast cells from a female Rongshui pig to precisely express mutant HTT at the endogenous level.

Figure 2. Schematic diagram of the strategy to generate HD pigs via homologous recombination.

A total of 2,430 fibroblast cells were screened and 9 were identified as positive cell clones with human HTT exon 1 inserted at the right locus. One of the positive clones was selected for somatic cell nuclear transfer (SCNT) and obtained 2,880 embryos, which were then transferred into 16 surrogate pigs and finally got 7 piglets naturally delivered. Through PCR analysis, 6 of them carried the human HTT exon 1 with expanded CAG repeats. The female founder (F0) pigs were used to hybridize with wild-type (WT) male Bama pigs to generate F1 pigs. After screening, knocked-in (KI) male F1 pigs were mated with WT female Bama pigs to generate F2 pigs. Over two years of breeding, 15 F1 pigs and 10 F2 pigs were yield, and fortunately, they were all carrying mutant HTT.

By delivering the Cas9 nuclease complexed with a synthetic gRNA into a cell, the cell’s genome can be cut at the desired location, allowing existing genes to be removed or replaced by new ones. However, the CRISPR/Cas9 mediated gene editing has a risk of off target, which can alter the function of otherwise intact genes. Once it occurs in the KI pigs, it will be hard to distinguish the pathological features are caused by the knocked-in genes or off-target activities. To solve this problem, the research team cooperated with Annoroad, conducted whole-genome sequencing upon genomic DNAs from the cortical tissues of F1 KI pigs. Most likely off-target loci by HTT gRNA1 and gRNA2 was checked, and the analysis shows no off-target mutations in the cortex of F1 KI pig.

Figure 3. off-target assay and whole genome-sequencing.

Then the characterization of the HD KI pigs was focused on F0 and F1 KI pigs to investigate their behavioral and pathological changes. HD KI pigs did not show obvious symptoms before the age of 4 months. By continuously monitoring they started showing some symptoms, including less body weight gain, wrinkled and sagging skin, abnormal walking pattern, respiratory difficulty, irregular breathing pattern and even earlier death. The breath difficulty suggests that respiratory failure could contribute to the death of animals and is consistent with the observation that pulmonary dysfunction and aspiration pneumonia/suffocation are the major cause of the death in HD patients.

Figure 4. Photo of a symptomatic HD KI pig (F0-5) and a WT pig at 5 months old, the foot-printing assay revealing gait abnormalities in the KI pig (F0-5) compared with the WT control.

The most important finding in this study is the presence of robust and selective neurodegeneration in the HD KI pig brains, which mimics the severe and preferential neurodegeneration of the medium spiny neurons in HD patients. Thus, the HD KI pigs are ideal models which share the typical behaviour and pathological features of HD patients. This research also paves an avenue for generating animal models to mimic selective neurodegeneration in other critical neurodegenerative diseases, such as AD and PD, and to develop effective therapeutic strategies.

The off-target assay and whole-genome sequencing (WGS) after CRISPR/Cas9 gene editing were conducted by Annoroad. We have launched topspeed WGS service, which only takes 8 working days to get data. Our high-throughput automated platform is applied to the library preparation for individual WGS, population evolution and GWAS, etc.

WGS for off-target loci uses Cas9 and other similar nucleases to break dsDNA and then test off-target loci using high-throughput sequencing method, which can find loci with mutation rates less than 0.1%. Annoroad can conduct comprehensive off-target assay to gene-editing cells or individuals via CRISPR-WGS, which provides a more quick and accurate way to accomplish and evaluate the CRISPR gene-editing technique.



Yan, S., et al., A Huntingtin Knockin Pig Model Recapitulates Features of Selective Neurodegeneration in Huntington’s Disease. Cell, 2018.


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