CRISPR/Cas9-mediated PINK1 deletion leads to neurodegeneration in rhesus monkeys

PINK1 mutations cause autosomal recessive and early-onset Parkinson’s disease (PD) with selective neurodegeneration. Unfortunately, current PINK1 knockout (KO) mouse 1 , 2 and pig models 3 , 4 are unable to recapitulate the selective and overt neurodegeneration seen in PD patients. Furthermore, endogenous Pink1 in the mouse brain is expressed at very low levels and can only be detected via immunoprecipitation, 5 meaning that PINK1’s function in the mammalian brain needs to be assessed using larger animals that are closer to humans. We previously used CRISPR/Cas9 to target the monkey gene in one-cell stage embryos. 6 Using the same approach, we designed two gRNAs to target exon 2 (T1) and exon 4 (T2), which encode a kinase domain in the PINK1 gene of rhesus monkeys (Fig. 1a ). CRISPR/Cas9 and PINK1 gRNAs were injected into one-cell stage rhesus monkey embryos. A T7E1 assay and sequencing of PCR products from the injected embryos showed high efficiency (61.5%) in targeting PINK1 (Fig. 1b and Supplementary information, Fig. S1 ). Transfer of 87 embryos to 28 surrogate rhesus monkeys resulted in 11 pregnancies (39.2%) (Fig. 1b ). Eleven fetuses developed to term and were born naturally. Of these live monkeys, eight carried PINK1 mutations (M), and three were wild type (WT). However, three mutant monkeys (M1, M3 and M4) were newborn triplets that struggled to survive and died 3-4 days after birth. One WT newborn monkey also died after a difficult labor. Another mutant monkey (M2) died 7 days after birth without noticeable warning signs or symptoms. The other three mutant monkeys (M6, M7 and M8) have lived for three years; M5, however, reduced its food intake and showed weakness at the age of 1.5 years, and died 30 days after anesthesia for MRI examination. Fig. 1 a PINK1 protein domains and targeted regions. MTS (mitochondrial targeting sequence), TMD (transmembrane domain), exon 2 (T2) and exon 4 (T4) targeting regions are indicated. b Summary of embryo injection, transfer, pregnancy, and newborn monkeys. c Diagram of PCR primers designed to determine the PINK1 large deletion. d Large PINK1 deletion in the cortex and striatum of M1, M2, M3, M4, and M5 monkeys. Exon 3 PINK1 represents the remaining intact PINK1 , whereas truncated PINK1 is generated by large deletion. e Western blot analysis of the brain cortical tissues of four PINK1 mutant monkeys and three newborn wild-type (WT) monkeys. The tissues were probed with antibodies to PINK1, neuronal proteins (NeuN, PSD95, CRMP2, and SNAP25), β-actin, and doublecortin (DCX). f Quantitative analysis of the ratio of exon 3 PINK1 or truncated PINK1 product to actin revealed that the cortex (Ctx) and striatum (Str) of M1 and the cortex of M2 had an extensive large deletion. The results were obtained from three PCR experiments. g Inverse correlation of the rate of the large PINK1 deletion (truncated PINK1/actin) with the relative levels of PINK1 (PINK1/actin) or NeuN (NeuN/actin) revealed by western blotting ( n = 3 independent experiments via analysis of the cortical tissues in three WT and four mutant monkeys). h Representative immunostaining micrographs show loss of NeuN-positive neurons in the cortex of PINK1 mutant monkeys (M1 and M2) compared with WT controls. Scale bar, 30 μm. i Quantitative assessment of NeuN-positive neurons in the cortex and striatum of newborn PINK1 mutant monkeys (M1 and M2) and two WT control monkeys. ** P < 0.05, *** P < 0.01. j Western blotting of lysates from WT or M1 monkey brain cortex and the human brain hippocampus. Red arrow indicates PINK1. k Western blot analysis of cortex and substantia nigra lysates of M6 monkey and age-matched 3-year-old WT monkey. Red arrow indicates PINK1. l Electron microscopy revealed degenerated cells in the cortex and substantia nigra of the M6 monkey. Degenerated neurons show electron-dense cytoplasm with no clear organelles and no identifiable nuclear membrane, and dark neurons are more obvious in the M6 substantia nigra. The enlarged micrographs beneath the corresponding WT and M6 images are from their boxed areas and show mitochondrial morphology. Scale bars, 2 μm Full size image