Summary Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

CRISPR/Cas9 is now widely used in biomedical research and has great potential for clinical applications. However, the safety and efficacy of this gene-editing technique are significant issues. Recent reports on mouse models and human cells have raised concerns that off-target mutations could hamper applying the CRISPR technology in patients. The high similarities of nonhuman primates to humans in genome content and organization, genetic diversity, physiology, and cognitive abilities have made these animals ideal experimental models for understanding human diseases and developing therapeutics. Off-target mutations of CRISPR/Cas9 have been analyzed in previous studies of nonhuman primates, but no report has investigated genome-wide off-target effects in living monkeys. Here, we used rhesus monkeys in which a genetic disorder mimicking Duchenne muscular dystrophy had previously been produced with CRISPR/Cas9. Using whole-genome sequencing to comprehensively assess on- and off-target mutations in these animals, we found that CRISPR/Cas9-based gene editing is active on the expected genomic sites without producing off-target modifications in other functional regions of the genome. These findings suggest that the CRISPR/Cas9 technique could be relatively safe and effective in modeling genetic disease in nonhuman primates and in future therapeutic research of human diseases.

Splice-switching antisense oligonucleotides (ASOs) and engineered U7 small nuclear ribonucleoprotein (U7 Sm OPT) are the most commonly used methods for exon skipping. However, challenges remain, such as limited organ delivery and repeated dosing for ASOs and unknown risks of by-products produced by U7 Sm OPT. Here, we showed that antisense circular RNAs (AS-circRNAs) can effectively mediate exon skipping in both minigene and endogenous transcripts. We also showed a relatively higher exon skipping efficiency at the tested Dmd minigene than U7 Sm OPT. AS-circRNA specifically targets the precursor mRNA splicing without off-target effects. Moreover, AS-circRNAs with adeno-associated virus (AAV) delivery corrected the open reading frame and restored the dystrophin expression in a mouse model of Duchenne muscular dystrophy. In conclusion, we develop an alternative method for regulating RNA splicing, which might be served as a novel tool for genetic disease treatment.

The tumor suppressor p53 is a key regulator of cell apoptosis and cell cycle arrest. Recent studies show that the delicate balance of p53 expression is important for neural tube defects, neuronal degeneration, embryonic lethality, as well as differentiation and dedifferentiation. Moreover, p53 showed different regulatory patterns between rodent and primate embryonic stem cells (ESCs). However, the role of p53 and apoptosis stimulating protein of p53 (ASPP) during neural differentiation (ND) from primate ESCs is still unknown. In this study, using an FGF-2 and/or HGF selectively containing ND culture systems for rhesus monkey ESCs (rESCs), the changes of p53 and ASPPs, and p53 targets, i.e. BAX and p21, were analyzed. Our results showed that the expression patterns of ASPP1/ASPP2 and iASPP were opposite in rESCs but similar in differentiated cells, and the expression of p53 was approximately consistent with BAX, but not p21. These findings indicate that the strong expression of iASPP in ESCs and weak expression of ASPP1/ASPP2 maintain the stability of stemness; and in ND niche, unimpaired iASPP may decrease its inhibition of ASPP1/ASPP2 expression, the interaction of p53 and ASPPs causing rESCs to convert towards a neural fate concomitant with apoptosis, but not to cell cycle arrest.

16The CRISPR-mediated Cas system is the most widely used tool in gene editing 17 and gene therapy for its convenience and efficiency. Delivery of the CRISPR system 18 by adeno-associated viruses (AAVs) is currently the most promising approach to gene 19 therapy. However, pre-existing adaptive immune responses against CRISPR nuclease 20 (PAIR-C) and AAVs has been found in human serum, indicating that immune response 21 is a problem that cannot be ignored, especially for in vivo gene correction. Non-human 22 primates (NHPs) share many genetic and physiological traits with human, and are 23 considered as the bridge for translational medicine. However, whether NHPs have same 24 PAIR-C status with human is still unknown. Here, macaques (rhesus and cynomolgus), 25 including normal housed and CRISPR-SpCas9 or TALENs edited individuals, were 26 used to detect PAIR-C which covered SaCas9, SpCas9, AsCas12a and LbCas12a. Dogs 27 and mice were also detected to expand the range of species. In addition, pre-existing 28 adaptive antibodies to AAV8 and AAV9 were performed against macaques of different 29 ages. The results showed that adaptive immunity was pre-existing in the macaques 30 regardless of Cas proteins and AAVs. These findings indicate that the pre-existing 31 adaptive immune of AAV-delivered CRISPR construction and correction system 32 should be concerned for in vivo experiments. 2 33 34 The existing popular gene editing technologies mainly include zinc finger nuclease 35 (ZFN), transcription activator-like effector nucleases (TALENs), and clustered regular 36 interspaced short palindromic repeats (CRISPR) [1-3]. Through these techniques, target 37 genes can be knocked out or knocked in, and animal models of diseases caused by gene 38 mutations can be obtained[4-7]. Due to its convenient design, simple operation and high 39 efficiency, CRISRP system has become the most used gene editing system[8]. With the 40 further development of the CRISPR system, it has begun to be applied to preclinical 41 trials to treat or repair hereditary diseases caused by gene mutations[9]. Currently, there 42 are two main methods for applying the CRISPR system to therapeutic preclinical 43 experiments. One is in vitro editing. For example, stem cells are edited in vitro, and the 44 repaired cells are returned to diseased tissues and organs[10-12]. The other is in vivo 45 editing, using vectors to deliver the CRISPR system to the diseased area by intravenous, 46 intramuscular or intraperitoneal injection[13-15]. However, due to the inherent nature 47 of the CRISPR system, its safety such as off-target[16-18], immune response[19-21] 48 and toxicity[22-24] have not been well resolved, making it a huge challenge for clinic 49 application. 50 The CRISPR system is mainly derived from bacteria and archaea [25], which often 51 invade organisms as pathogens, and are recognized as an alien by the immune system 52 of these organisms, activates lymphocytes in the body to produce corresponding 53 effector cells, and removes them[26]. It is difficult f...

IntroductionPolycystic kidney disease (PKD) is a common autosomal dominant or recessive genetic disease, often accompanied by polycystic liver disease (PLD). Many cases of PKD in animals have been reported. However, little is known about the genes that cause PKD in animals.MethodsIn this study, we evaluated the clinical phenotypes of PKD in two spontaneously aged cynomolgus monkeys and explored the genetic etiology using whole-genome sequencing (WGS). Ultrasonic and histological consequences were further investigated in PKD- and PLD-affected monkeys.ResultsThe results indicated that the kidneys of the two monkeys had varying degrees of cystic changes, and the renal cortex was thinned and accompanied by fluid accumulation. As for hepatopathy, inflammatory cell infiltration, cystic effusion, steatosis of hepatocytes, and pseudo-lobular were found. Based on WGS results, the variants of PKD1:(XM_015442355: c.1144G>C p. E382Q) and GANAB: (NM_001285075.1: c.2708T>C/p. V903A) are predicted to be likely pathogenic heterozygous mutations in PKD- and PLD-affected monkeys.DiscussionOur study suggests that the cynomolgus monkey PKD and PLD phenotypes are very similar to those in humans, and are probably caused by pathogenic genes homologous to humans. The results indicate that cynomolgus monkeys can be used as the most appropriate animal model for human PKD pathogenesis research and therapeutic drug screening.

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Background The CRISPR/Cas9 system can induce off-target effects in programmed gene editing, but there have been few reports on cleavage detection and characterization in early embryos. To study these events, sgRNAs with different off-target rates were designed by off-target prediction software and injected into mouse zygotes with Cas9. The development of the injected embryos was analyzed, and γH2AX, which can bind sequences at double-strand breaks (DSBs), was used for DNA cleavage analysis by immunostaining and CUT&Tag. Results The results showed that the sgRNAs with a higher off-target rate were associated with significantly reduced blastocyst rate and birth rate in injected mouse embryos. γH2AX immunofluorescence indicated that there was a relative DSB peak at 15 h after Cas9 system injection, and the number of γH2AX foci at the peak was significantly higher in the low off-target sgRNA-injected group (101.3 ± 16.6) than in the control group (42.2 ± 9.9). No predicted off-target mutations with a difference of 4 bases from the sgRNA were found in the Cas9-edited mice or fetuses. At 15 h after CRISPR/Cas9 and low off-target sgRNA injection, many sgRNA-independent DSBs were detected by CUT&Tag sequencing. The distribution of sgRNA-independent DSBs had no chromosome specificity. Gene Ontology (GO) annotation analysis of the sgRNA-independent DSB sites showed that the sgRNA-independent DSBs were mainly concentrated at genes associated with biological processes such as neuron projection morphogenesis and steroid hormone-mediated signaling; molecular functions such as PDZ domain binding and actin binding; and cell components such as postsynaptic membrane and intracellular vessel. Conclusion In conclusion, the many sgRNA-independent cleavages induced when the Cas9 system is used for gene editing in mouse embryos were visually detected and characterized in the genome for the first time. These results should also be considered when using or optimizing the Cas9 system.