Precise therapeutic gene correction by a simple nuclease-induced double-stranded break

Iyer, Sukanya; Suresh, Sneha; Guo, Dongsheng; Daman, Katelyn; Chen, Jennifer C. J.; Liu, Pengpeng; Zieger, Marina; Luk, Kevin; Roscoe, Benjamin P.; Mueller, Christian; King, Oliver D.; Emerson, Charles P.; Wolfe, Scot A.

doi:10.1038/s41586-019-1076-8

Cited by 94 publications

(74 citation statements)

References 46 publications

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“…HPS pathogenesis and therapeutic options continue to be investigated, including through the use of organoids (Korogi et al, 2019; Strikoudis et al, 2019) and explorations of gene therapy (Ikawa et al, 2015; Iyer et al, 2019; Shen et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Hermansky–Pudlak syndrome: Mutation update

et al. 2020

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Hermansky-Pudlak syndrome (HPS) is a group of 10 autosomal recessive multisystem disorders, each defined by the deficiency of a specific gene. HPS-associated genes encode components of four ubiquitously expressed protein complexes: Adaptor protein-3 (AP-3) and biogenesis of lysosome-related organelles complex-1 (BLOC-1) through -3. All individuals with HPS exhibit albinism and a bleeding diathesis; additional features occur depending on the defective protein complex. Pulmonary fibrosis is associated with AP-3 and BLOC-3 deficiency, immunodeficiency with AP-3 defects, and gastrointestinal symptoms are more prevalent and severe in BLOC-3 deficiency. Therefore, identification of the HPS subtype is valuable for prognosis, clinical management, and treatment options. The prevalence of HPS is estimated at 1-9 per 1,000,000. Here we summarize 264 reported and novel variants in 10 HPS genes and estimate that~333 Puerto Rican HPS subjects and~385 with other ethnicities are reported to date. We provide pathogenicity predictions for missense and splice site variants and list variants with high minor allele frequencies. Current cellular and clinical aspects of HPS are also summarized. This review can serve as a manifest for molecular diagnostics and genetic counseling aspects of HPS.

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Section: Introductionmentioning

confidence: 99%

Hermansky–Pudlak syndrome: Mutation update

et al. 2020

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“…Taken together, our results further emphasize the potential founder effect of this mutation in East and South Asia. A recent study focusing on this TCAP mutation demonstrated promising therapeutic results by gene editing using patient-derived induced pluripotent stem cells, highlighting the importance of accurate molecular diagnosis for patients with hereditary muscle diseases [2].…”

Section: Discussionmentioning

confidence: 99%

“…Currently, LGMD, divided into type 1 and 2 depending on autosomal dominant or recessive inheritance, comprises more than 30 subtypes. Clinical, pathological, or genetic analysis in isolation is typically insufficient for precise diagnosis; multidisciplinary evaluation in conjunction with genetic testing is required to achieve final diagnosis and essential for outcome prediction, comorbidity prevention, genetic counseling, and further precise mRNA-targeting or gene correction therapy [2].…”

Section: Introductionmentioning

confidence: 99%

Clinical, pathological, imaging, and genetic characterization in a Taiwanese cohort with limb-girdle muscular dystrophy

Liang

Jong

Wang

et al. 2020

Orphanet J Rare Dis

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Background: Limb-girdle muscular dystrophy (LGMD) is a genetically heterogeneous, hereditary disease characterized by limb-girdle weakness and histologically dystrophic changes. The prevalence of each subtype of LGMD varies among different ethnic populations. This study for the first time analyzed the phenotypes and genotypes in Taiwanese patients with LGMD in a referral center for neuromuscular diseases (NMDs). Results: We enrolled 102 patients clinically suspected of having LGMD who underwent muscle biopsy with subsequent genetic analysis in the previous 10 years. On the basis of different pathological categories, we performed sequencing of target genes or panel for NMDs and then identified patients with type 1B, 1E, 2A, 2B, 2D, 2I, 2G, 2 N, and 2Q. The 1B patients with LMNA mutation presented with mild limb-girdle weakness but no conduction defect at the time. All 1E patients with DES mutation exhibited predominantly proximal weakness along with distal weakness. In our cohort, 2B and 2I were the most frequent forms of LGMD; several common or founder mutations were identified, including c.1097_1099delACA (p.Asn366del) in DES, homozygous c.101G > T (p.Arg34Leu) in SGCA, homozygous c.26_33dup (p.Glu12Argfs*20) in TCAP, c.545A > G (p.Tyr182Cys), and c.948delC (p.Cys317Alafs*111) in FKRP. Clinically, the prevalence of dilated cardiomyopathy in our patients with LGMD2I aged > 18 years was 100%, much higher than that in European cohorts. The only patient with LGMD2Q with PLEC mutation did not exhibit skin lesions or gastrointestinal abnormalities but had mild facial weakness. Muscle imaging of LGMD1E and 2G revealed a more uniform involvement than did other LGMD types. Conclusion: Our study revealed that detailed clinical manifestation together with muscle pathology and imaging remain critical in guiding further molecular analyses and are crucial for establishing genotype-phenotype correlations. We also determined the common mutations and prevalence for different subtypes of LGMD in our cohort, which could be useful when providing specific care and personalized therapy to patients with LGMD.

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“…Protein purification for 3xNLS-SpyCas9 and Cas12a-2xNLS proteins followed a common protocol as previously described. 66 The generation and characterization of the 3xNLS-SpyCas9 and LbaCas12a-2xNLS constructs have been recently described. 52, 67, 68 The pET21a plasmid backbone (Novagen) was used to drive the expression of a hexa-His-tagged version of each protein.…”

Section: Methodsmentioning

confidence: 99%

Efficient Homology-directed Repair with Circular ssDNA Donors

Iyer

Mir

Vega-Badillo

et al. 2019

Preprint

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32While genome editing has been revolutionized by the advent of CRISPR-based 33 nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair 34(HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer 35 from low HDR efficiencies in many cell types, as well as integration at unintended sites. 36In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with 37 minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently 38 and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors 39 serve as efficient HDR templates when used with Cas9 or Cas12a, with integration 40 frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative 41 efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a 42 nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-43

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Precise therapeutic gene correction by a simple nuclease-induced double-stranded break

Cited by 94 publications

References 46 publications

Hermansky–Pudlak syndrome: Mutation update

Hermansky–Pudlak syndrome: Mutation update

Clinical, pathological, imaging, and genetic characterization in a Taiwanese cohort with limb-girdle muscular dystrophy

Efficient Homology-directed Repair with Circular ssDNA Donors

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