Multiplex precise base editing in cynomolgus monkeys.

Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity of base editing in monkey embryos. Our data demonstrate feasibility of multiplex base editing for polygenic disease modeling in primate zygotes.

Suppression of Bone Resorption by miR-141 in Aged Rhesus Monkeys.

Aging-related osteoporosis (OP) is considered a serious public health concern. Approximately 30% of postmenopausal women suffer from OP; more than 40% of them risk fragility fractures. Multiple drugs have been prescribed to treat OP, but they are not ideal because of low cure rates and adverse side effects. miRNA-based gene therapy is a rapidly developing strategy in disease treatment that presents certain advantages, such as large-scale production capability, genetic safety, and rapid effects. miRNA drugs have been used primarily in cancer treatments; they have not yet been reported as candidates for osteoclast-targeted-OP treatment in primates. Their therapeutic efficacy has been limited by several shortcomings, such as low efficiency of selective delivery, insufficient expression levels in targeting cells, and unexpected side effects. Here, we identify miR-141 as a critical suppressor of osteoclastogenesis and bone resorption. The expression levels of miR-141 are positively correlated with BMD and negatively correlated with the aging of bones in both aged rhesus monkeys (Macaca mulatta) and osteoporotic patients. Selective delivery of miR-141 into the osteoclasts of aged rhesus monkeys via a nucleic acid delivery system allowed for a gradual increase in bone mass without significant effects on the health and function of primary organs. Furthermore, we found that the functional mechanism of miR-141 resides in its targeting of two osteoclast differentiation players, Calcr (calcitonin receptors) and EphA2 (ephrin type-A receptor 2 precursor). Our study suggests that miRNAs, such as miR-141, could play a crucial role in suppressing bone resorption in primates and provide reliable experimental evidence for the clinical application of miRNA in OP treatment. © 2018 American Society for Bone and Mineral Research.

Efficient production of cynomolgus monkeys with a toolbox of enhanced assisted reproductive technologies.

The efficiency of assisted reproductive technologies (ARTs) in nonhuman primates is low due to no screening criterions for selecting sperm, oocyte, and embryo as well as its surrogate mothers. Here we analyzed 15 pairs of pregnant and non-pregnant cynomolgus monkeys, each pair of which received embryos from one batch of fertilized oocytes, and found ratio of endometrial to myometrial thicknesses in abdominal ultrasonic transverse section of uterus is a reliable indicator for selection of recipients for embryo transfer. We performed 305 ovarian stimulations in 128 female cynomolgus monkeys and found that ovarian stimulation can be performed in a whole year and repeated up to six times in the same monkey without deteriorating fertilization potential of eggs until a poor response to stimulation happened. Fertilization can be efficiently achieved with both conventional and piezo-driven intracytoplasmic sperm injection procedures. In semen collection, semen quality is higher with the penile robe electrical stimulus method compared with the rectal probe method. Moreover, caesarean section is an effective strategy for increasing baby survival rates of multiple pregnancies. These findings provide a practical guidance for the efficient use of ARTs, facilitating their use in genetic engineering of macaque monkeys for basic and translational neuroscience research.

Off-target Effects in CRISPR/Cas9-mediated Genome Engineering.

CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)-RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site-is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype-phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.