CRISPR applications for Duchenne muscular dystrophy: From animal models to potential therapies.

CRISPR gene-editing technology creates precise and permanent modifications to DNA. It has significantly advanced our ability to generate animal disease models for use in biomedical research and also has potential to revolutionize the treatment of genetic disorders. Duchenne muscular dystrophy (DMD) is a monogenic muscle-wasting disease that could potentially benefit from the development of CRISPR therapy. It is commonly associated with mutations that disrupt the reading frame of the DMD gene that encodes dystrophin, an essential scaffolding protein that stabilizes striated muscles and protects them from contractile-induced damage. CRISPR enables the rapid generation of various animal models harboring mutations that closely simulates the wide variety of mutations observed in DMD patients. These models provide a platform for the testing of sequence-specific interventions like CRISPR therapy that aim to reframe or skip DMD mutations to restore functional dystrophin expression. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics.

Antisense oligonucleotide therapy for KCNT1 encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are characterized by pharmaco-resistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, which encodes for sodium activated potassium channel protein KNa1.1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene-silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared with mice treated with a control ASO (nonhybridizing sequence). ASO administration at neonatal age was also well tolerated and effective in controlling seizures and extending the life span of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.

The Nestin neural enhancer is essential for normal levels of endogenous Nestin in neuroprogenitors but is not required for embryo development.

Enhancers are vitally important during embryonic development to control the spatial and temporal expression of genes. Recently, large scale genome projects have identified a vast number of putative developmental regulatory elements. However, the proportion of these that have been functionally assessed is relatively low. While enhancers have traditionally been studied using reporter assays, this approach does not characterise their contribution to endogenous gene expression. We have studied the murine Nestin (Nes) intron 2 enhancer, which is widely used to direct exogenous gene expression within neural progenitor cells in cultured cells and in vivo. We generated CRISPR deletions of the enhancer region in mice and assessed their impact on Nes expression during embryonic development. Loss of the Nes neural enhancer significantly reduced Nes expression in the developing CNS by as much as 82%. By assessing NES protein localization, we also show that this enhancer region contains repressor element(s) that inhibit Nes expression within the vasculature. Previous reports have stated that Nes is an essential gene, and its loss causes embryonic lethality. We also generated 2 independent Nes null lines and show that both develop without any obvious phenotypic effects. Finally, through crossing of null and enhancer deletion mice we provide evidence of trans-chromosomal interaction of the Nes enhancer and promoter.

Optimized nickase- and nuclease-based prime editing in human and mouse cells.

Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency.

Progress Toward Zygotic and Germline Gene Drives in Mice.

CRISPR-based synthetic gene drives have the potential to deliver a more effective and humane method of invasive vertebrate pest control than current strategies. Relatively efficient CRISPR gene drive systems have been developed in insects and yeast but not in mammals. Here, we investigated the efficiency of CRISPR-Cas9-based gene drives in Mus musculus by constructing "split drive" systems where gRNA expression occurs on a separate chromosome to Cas9, which is under the control of either a zygotic (CAG) or germline (Vasa) promoter. While both systems generated double-strand breaks at their intended target site in vivo, no homology-directed repair between chromosomes ("homing") was detectable. Our data indicate that robust and specific Cas9 expression during meiosis is a critical requirement for the generation of efficient CRISPR-based synthetic gene drives in rodents.

A Y-chromosome shredding gene drive for controlling pest vertebrate populations.

Self-replicating gene drives that modify sex ratios or infer a fitness cost could be used to control populations of invasive alien species. The targeted deletion of Y sex chromosomes using CRISPR technology offers a new approach for sex bias that could be incorporated within gene-drive designs. We introduce a novel gene-drive strategy termed Y-CHromosome deletion using Orthogonal Programmable Endonucleases (Y-CHOPE), incorporating a programmable endonuclease that 'shreds' the Y chromosome, thereby converting XY males into fertile XO females. Firstly, we demonstrate that the CRISPR/Cas12a system can eliminate the Y chromosome in embryonic stem cells with high efficiency (c. 90%). Next, using stochastic, individual-based models of a pest mouse population, we show that a Y-shredding drive that progressively depletes the pool of XY males could effect population eradication through mate limitation. Our molecular and modeling data suggest that a Y-CHOPE gene drive could be a viable tool for vertebrate pest control.

Expanding the RNA-Guided Endonuclease Toolkit for Mouse Genome Editing.

The RNA-guided endonuclease CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is widely used for generating genetically modified mice via zygotic microinjection. Although SpCas9 is a potent mutagen, it requires an NGG proto-spacer adjacent motif (PAM) at the target site, restricting sequence targetability. Here, we show that RNA-guided endonucleases that utilize a range of alternative PAM sequences can edit the mouse genome at the neurog3 (Ngn3) locus: SpCas9 VQR (NGAN PAM), SpCas9 VRER (NGCG), AsCas12a (TTTN), SaCas9 (NNGRRT), and SaCas9 KKH (NNNRRT). Additional experiments targeting tyrosinase and frizzled3 with SaCas9 KKH and its parent protein demonstrated that these endonucleases generated mutations in up to 100% of embryos across three loci. Remarkably, in contrast to wild-type SpCas9, these endonucleases frequently generated mutant embryos that retain unmodified alleles in both template-free and HDR-repair experiments. Our findings broaden PAM recognition options for mouse genome editing and identify SaCas9/SaCas9 KKH as useful alternatives when targeting genes with null lethal phenotypes.

Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression.

CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs. Through nucleofection of mouse embryonic stem cells, we demonstrate highly efficient cleavage of the target loci using the dual-guide plasmids, which are available as Cas9-nuclease or Cas9-nickase expression constructs, with or without selection markers. These vectors are a valuable addition to the CRISPR/Cas9 toolbox and will be made available to all interested researchers via the Addgene plasmid repository.

Viperin is an important host restriction factor in control of Zika virus infection.

Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-ß and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin-/- mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.

Targeted Deletion of an Entire Chromosome Using CRISPR/Cas9.

CRISPR/Cas9 genome editing can facilitate efficient deletion of genomic region, but it has not been used to delete an entire chromosome. Here, Adikusuma et al. show proof-of-concept for efficient CRISPR-mediated selective chromosome deletion by removing the centromere or shredding the chromosome arm in mouse embryonic stem cells and zygotes.

Functional Equivalence of the SOX2 and SOX3 Transcription Factors in the Developing Mouse Brain and Testes.

Gene duplication provides spare genetic material that evolution can craft into new functions. Sox2 and Sox3 are evolutionarily related genes with overlapping and unique sites of expression during embryogenesis. It is currently unclear whether SOX2 and SOX3 have identical or different functions. Here, we use CRISPR/Cas9-assisted mutagenesis to perform a gene-swap, replacing the Sox3 ORF with the Sox2 ORF to investigate their functional equivalence in the brain and testes. We show that increased expression of SOX2 can functionally replace SOX3 in the development of the infundibular recess/ventral diencephalon, and largely rescues pituitary gland defects that occur in Sox3 null mice. We also show that ectopic expression of SOX2 in the testes functionally rescues the spermatogenic defect of Sox3 null mice, and restores gene expression to near normal levels. Together, these in vivo data provide strong evidence that SOX2 and SOX3 proteins are functionally equivalent.