Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9

Abstract

CRISPR/Cas9 has been used to genetically modify genomes in a variety of species, including non-human primates. Unfortunately, this new technology does cause mosaic mutations, and we do not yet know whether such mutations can functionally disrupt the targeted gene or cause the pathology seen in human disease. Addressing these issues is necessary if we are to generate large animal models of human diseases using CRISPR/Cas9. Here we used CRISPR/Cas9 to target the monkey dystrophin gene to create mutations that lead to Duchenne muscular dystrophy (DMD), a recessive X-linked form of muscular dystrophy. Examination of the relative targeting rate revealed that Crispr/Cas9 targeting could lead to mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle. Moreover, CRISPR/Cas9 induced mutations in both male and female monkeys, with the markedly depleted dystrophin and muscle degeneration seen in early DMD. Our findings indicate that CRISPR/Cas9 can efficiently generate monkey models of human diseases, regardless of inheritance patterns. The presence of degenerated muscle cells in newborn Cas9-targeted monkeys suggests that therapeutic interventions at the early disease stage may be effective at alleviating the myopathy.

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

“…[16][17][18] Recent reports have indicated that the CRISPR/Cas9 system can be used for genetic modification in NHPs; however, this approach has not yet been applied to insert genes of interest into the AAVS1 safe harbor in NHP PSCs. [19][20][21] We have identified a CRISPR/Cas9 target sequence in the rhesus macaque PPP1R12C gene, generated safe harbor-targeted rhesus macaque iPSC (RhiPSC) lines with two relevant marker genes, and documented the stability of expression both in iPSCs and after differentiation to tissues of interest. Genes encoding human truncated CD19 (hDCD19) or GFP were chosen as reporters.…”

Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery

“…However, HDR rates can be increased in some cell types through cell cycle synchronization 40 or the use of small molecules or proteins that interfere with alternate DNA-repair pathways 44,45 . In sum, Cas9-based technological advances have dramatically simplified the creation of modified vertebrate cell lines and animal models for the investigation of gene function during development and disease progression 25,38,[46][47][48][49][50][51][52][53][54][55][56] . A similar revolution is being realized by the use of nuclease-dead Cas9 (dCas9) to deliver effector domains to perturb gene expression and chromatin-modification states 57 .…”

Using Genome Engineering to Understand Huntington’s Disease

“…The modified Cas9n was found to have similar cleavage efficiency when two gRNAs were used, one targeted on each strand of the DNA, resulting in a double strand break. The technique has been widely adopted to create disease-modeling cell lines, rodent and non-human primate models and in non-viable human embryos (Liang et al 2015;Chen et al 2015). What has made the CRISPR system so accessible is that, unlike the zinc fingers and TALEs, the same core protein, Cas9, is used to target any sequence, whereas the targeting portion of the CRISPR system, the gRNA, is what varies.…”