CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model.

BACKGROUND: The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. RESULTS: To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout. CONCLUSIONS: Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.

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.

Killing two birds with one stone: CRISPR/Cas9 CCR5 knockout hematopoietic stem cells transplantation to treat patients with HIV infection and hematological malignancies concurrently.

Human immunodeficiency virus (HIV) is known to cause hematological malignancy. Hematopoietic stem cell transplantation (HPSCT) is an advanced treatment for that. Currently, there are three successful HIV-eliminated cases, and two received HPSCT from CCR5-absent donors. It is well established that the CCR5 protein on the cell surface assists human immunodeficiency virus entry. Preliminary studies have revealed that knocking out CCR5 and/or CXCR4 may inhibit the viral entry of HIV, which may prove promising in the further development of HIV treatment options. Herein, we suggest performing autologous or allogeneic HSCT with CCR5 KO hematopoietic stem cells in patients who suffer from complicated HIV conditions, particularly drug-resistant HIV or a concurrent diagnosis of HIV with lymphoma/leukemia, to achieve complete HIV remission. Nevertheless, at the clinical forefront of CRISPR-HIV technology, more efforts should be directed to advance nonhuman primate (NHP) models for studies of HIV pathogenesis and off-target assessments within this system. CRISPR-Cas9 knock out of host HSCT-expressing CCR5 or CXCR4 may confer HIV-resistance, which when applied to bedside therapeutics in an allogeneic or autologous manner can warrant a permanent and effective treatment outcome.

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.