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| Open AccessEnhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways
Precise genome editing is crucial. Here the authors demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor (CBE) or prime editor (PE) additively enhanced editing efficiency in hPSCs.
- Ju-Chan Park
- , Yun-Jeong Kim
- & Hyuk-Jin Cha
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Article
| Open AccessIn vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment
Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drug. Here, the authors show a promising CRISPR/Cas9 mediated homology-independent targeted integration therapy in patient derived cells and humanized mice carrying BCD mutations.
- Xiang Meng
- , Ruixuan Jia
- & Liping Yang
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Article
| Open AccessAn adeno-associated virus variant enabling efficient ocular-directed gene delivery across species
In non-human primates, rAAVs are delivered through suprachoroidal injection at a high dose to achieve optimal efficacy. Here, the authors present a novel AAV capsid (AAVv128) that significantly improved the transduction efficiency in photoreceptor and retinal pigment epithelial cells across species.
- Shuang Luo
- , Hao Jiang
- & Xun Sun
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Article
| Open AccessCoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing
Cas9 nucleases hold clinical significance for genome editing therapies. Here the authors characterize CoCas9, a compact, efficient and precise Cas9 from the human microbiome, and show that delivery via AAV vectors enables efficient editing in the mouse retina, expanding the genome editing toolbox.
- Eleonora Pedrazzoli
- , Michele Demozzi
- & Anna Cereseto
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Article
| Open AccessTranslation velocity determines the efficacy of engineered suppressor tRNAs on pathogenic nonsense mutations
An emerging therapeutic strategy is to suppress nonsense mutations with engineered suppressor tRNAs. Here, the authors show that the mRNA translation velocity is a key parameter determining the efficacy of suppressor tRNAs.
- Nikhil Bharti
- , Leonardo Santos
- & Zoya Ignatova
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Article
| Open AccessFunctional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair
Here the authors describe a functional screening platform in human stem cells to identify and optimize protein-based gene editing additives that increase homologous directed recombination and have potential to improve gene therapy workflows.
- Juan A. Perez-Bermejo
- , Oghene Efagene
- & Kristen L. Seim
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Article
| Open AccessHarnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation
Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important. This study introduces human liver ex situ normothermic perfusion as a model to evaluate gene therapy vectors, paving the way for advanced liver disease treatment.
- Marti Cabanes-Creus
- , Sophia H. Y. Liao
- & Leszek Lisowski
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Article
| Open AccessEnhancing prime editor activity by directed protein evolution in yeast
Compared to traditional Cas9 nucleases prime editors (PEs) are less active. Here the authors use OrthoRep, a yeast-based platform for directed protein evolution to enhance the editing efficiency of PEs: they identify mutations that have a positive effect on kinetics and use this knowledge to generate an efficient in vivo PE.
- Yanik Weber
- , Desirée Böck
- & Gerald Schwank
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Article
| Open AccessA humanized mouse model for adeno-associated viral gene therapy
All natural AAV serotypes transduce murine hepatocytes more efficiently than their human counterparts in human liver chimeric mouse models. Here the authors developed a novel humanized mouse were human transduction of AAV can be studied.
- Mercedes Barzi
- , Tong Chen
- & Karl-Dimiter Bissig
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Article
| Open AccessPhage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference
Existing TadA-derived CBEs exhibit residual A•T-to-G•C editing activity and suffer from lower activity at several sequence contexts and with non-SpCas9 targeting domains. Here, the authors use phage-assisted evolution to evolve CBE6 variants that address these limitations.
- Emily Zhang
- , Monica E. Neugebauer
- & David R. Liu
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Article
| Open AccessDomain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope
Nme2Cas9 has been well established as a genome editing platform. Here the authors engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors and validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.
- Nathan Bamidele
- , Han Zhang
- & Erik J. Sontheimer
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Article
| Open AccessCompact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells
The most recent class of base editors utilize DddAtox, a deaminase domain that can act upon double-stranded DNA. Here the authors target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers; they also identify a variety of DddAtox orthologues.
- Friedrich Fauser
- , Bhakti N. Kadam
- & Jeffrey C. Miller
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Article
| Open AccessAAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation
Long-term control of therapeutic transgene expression is needed. Here the authors report a muscone-induced transgene system packaged into AAVs based on a G protein-coupled murine olfactory receptor and a synthetic cAMP-responsive promoter: they show dose- and exposure-time-dependent gene expression control in mice.
- Xin Wu
- , Yuanhuan Yu
- & Haifeng Ye
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Article
| Open AccessTransient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells
Here the authors demonstrate that the frequency of HDR in human hematopoietic stem and progenitor cells is increased by the delivery of an inhibitor of 53BP1 as a recombinant peptide. This approach is applicable for a variety of therapeutically relevant loci in HSPCs as well in other primary human cell types.
- Ron Baik
- , M. Kyle Cromer
- & Matthew H. Porteus
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Article
| Open AccessDual targeted extracellular vesicles regulate oncogenic genes in advanced pancreatic cancer
KRASG12D mutations frequently co-occur with mutated TP53 tumour suppressor in patients with pancreatic ductal adenocarcinoma (PDAC). Here the authors report the design of dual targeted therapeutic extracellular vesicles containing high copy numbers of TP53 mRNA and siKRASG12D, showing anti-tumor activity in PDAC preclinical models.
- Chi-Ling Chiang
- , Yifan Ma
- & L. James Lee
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Article
| Open AccessmRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy
Large genes require dual adeno-associated viral (AAV) vectors for in vivo delivery/expression, but current methods have limitations. Here the authors develop and functionally evaluate REVeRT, an efficient and flexible dual AAV vector technology based on reconstitution via mRNA trans-splicing.
- Lisa Maria Riedmayr
- , Klara Sonnie Hinrichsmeyer
- & Elvir Becirovic
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Article
| Open AccessPAM-flexible genome editing with an engineered chimeric Cas9
CRISPR enzymes require a defined protospacer adjacent motif (PAM) which can be limiting for editing applications. Here the authors recombine the PAM-interacting domain of SpRY with the N-terminus of Sc + + to generate a chimeric enzyme with highly flexible PAM preference: SpRYc.
- Lin Zhao
- , Sabrina R. T. Koseki
- & Pranam Chatterjee
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Article
| Open AccessA Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo
SpCas9 unintended editing is a major concern. Here the authors report an off-target method using Duplex Sequencing with increased sensitivity for Cas9 mutation detection; they also identify a Cas9 variant of the II-B subfamily with intrinsic high fidelity (PsCas9) and see improved specificity.
- Burcu Bestas
- , Sandra Wimberger
- & Marcello Maresca
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Article
| Open AccessWhole genomic analysis reveals atypical non-homologous off-target large structural variants induced by CRISPR-Cas9-mediated genome editing
The safety of CRISPR-Cas9 editing is a concern. Here the authors use whole genomic analysis by 10x linked-read sequencing and optical genome mapping to interrogate the genome integrity after editing: they see large structural variants at on-target sites and unexpected large chromosomal deletions.
- Hsiu-Hui Tsai
- , Hsiao-Jung Kao
- & John Yu
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Article
| Open AccessAAV-mediated base-editing therapy ameliorates the disease phenotypes in a mouse model of retinitis pigmentosa
Base editing technology has great potential in treating pathogenic single-nucleotide variations. Using a dual-AAV base editing system, Wu et al. restored visual functions in a mouse model of retinitis pigmentosa.
- Yidong Wu
- , Xiaoling Wan
- & Xueli Zhang
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Article
| Open AccessmRNA lipid nanoparticle-mediated pyroptosis sensitizes immunologically cold tumors to checkpoint immunotherapy
mRNA nanomedicine-based gene therapy may offer opportunities for cancer treatment. Here the authors show that mRNA lipid nanoparticles encoding the N-terminal domain of gasdermin B trigger pyroptosis and promote anti-tumor immune responses in preclinical cancer models.
- Fengqiao Li
- , Xue-Qing Zhang
- & Xiaoyang Xu
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Article
| Open AccessTransplanting FVIII/ET3-secreting cells in fetal sheep increases FVIII levels long-term without inducing immunity or toxicity
The authors show in a preclinical large animal model that in utero delivery of FVIII-secreting human placental cells is feasible, safe, and yields elevated plasma FVIII activity for at least three years without triggering immunity, affirming prenatal treatment of hemophilia A.
- Martin Rodriguez
- , Brady Trevisan
- & Graça Almeida-Porada
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Article
| Open AccessCell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9
ON-target genotoxicity in gene editing is generally underestimated. Here the authors report Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems to detect and characterize rare large loss of heterozygosity: they show that ON-target genotoxicity can be prevented by p53 and cell cycle arrest.
- G. Cullot
- , J. Boutin
- & A. Bedel
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Article
| Open AccessRapid and definitive treatment of phenylketonuria in variant-humanized mice with corrective editing
The PAH P281L variant is one of the most common variants identified in phenylketonuria (PKU) patients. Here, the authors use base editing, enabled by lipid nanoparticle/mRNA technology, to directly correct the P281L variant in the liver in PKU mice and definitively treat the disease within 2 days.
- Dominique L. Brooks
- , Manuel J. Carrasco
- & Xiao Wang
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Article
| Open AccessTemplate-jumping prime editing enables large insertion and exon rewriting in vivo
Retrotransposons replicate their genetic information through target-primed reverse transcription (TPRT). Here the authors report a template-jumping prime editor (TJ-PE) to act similarly to TPRT and achieve insertions of large DNA fragments at endogenous sites: they rewrite a mutated exon in the mouse liver.
- Chunwei Zheng
- , Bin Liu
- & Wen Xue
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Article
| Open AccessDurable contraception in the female domestic cat using viral-vectored delivery of a feline anti-Müllerian hormone transgene
This study demonstrates the safety and long-term efficacy of a single-dose, injectable contraceptive in female domestic cats. Treated females remained contracepted for over two years, and did not ovulate or produce kittens when paired with males.
- Lindsey M. Vansandt
- , Marie-Charlotte Meinsohn
- & David Pépin
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Article
| Open AccessHematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy
Scala et al. show that mobilized peripheral blood hematopoietic stem/progenitor cells are more enriched in repopulating stem cells than bone marrow. Moreover, the quantity and type of infused subsets correlated with gene therapy outcome in humans.
- Serena Scala
- , Francesca Ferrua
- & Alessandro Aiuti
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Article
| Open AccessGalNAc-Lipid nanoparticles enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy
Kasiewicz et al. describe a structure-guided rational design approach to optimize a new GalNAc-Lipid nanoparticle that enables delivery of a base editing therapy in both low-density lipoprotein receptor-deficient and wild-type nonclinical models.
- Lisa N. Kasiewicz
- , Souvik Biswas
- & Andrew M. Bellinger
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Article
| Open AccessThe AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner
rAAV vectors vary in their effectiveness between species, making it difficult to predict clinical outcomes. Here authors show that AAV capsid proteins influence the vector epigenomic state in cells, and that a single amino acid change in the vector can alter the vector epigenome and hence transgene expression levels between species.
- Adriana Gonzalez-Sandoval
- , Katja Pekrun
- & Mark A. Kay
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Article
| Open AccessEngineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing
Cas12f proteins are small and sought after for therapeutic applications. Here the authors report six bacterial Cas12f1 proteins with nuclease activity in mammalian cells, perform sgRNA and protein engineering to generate variants with enhanced editing and broader PAMs, apply an inducible version in vivo.
- Xiangfeng Kong
- , Hainan Zhang
- & Hui Yang
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Article
| Open AccessRNAi-mediated rheostat for dynamic control of AAV-delivered transgenes
Here the authors propose an RNA interference-based switch for dynamic control of AAV transgene expression. In this approach, transgene expression may be silenced by RNAi and subsequently recovered using REVERSIR oligonucleotides.
- Megha Subramanian
- , James McIninch
- & Vasant Jadhav
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Article
| Open AccessLimitations of gene editing assessments in human preimplantation embryos
DNA repair in response to DSBs in the preimplantation embryo is hard to analyze. Here the authors show that over 25% of pre-existing heterozygous loci in control single blastomere samples appeared as homozygous after whole genome amplification, therefore, they validated gene editing seen in human embryos in ESCs.
- Dan Liang
- , Aleksei Mikhalchenko
- & Shoukhrat Mitalipov
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Review Article
| Open AccessAssessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing
CRISPR-Cas tools have shown exceptional promise in genome engineering over the past decade. Here the authors review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, as well as their editing precision, off-target effects, and clinical considerations.
- Jianli Tao
- , Daniel E. Bauer
- & Roberto Chiarle
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Article
| Open AccessSafeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration
Undesired chromosomal translocations, vector integrations, and large deletions remain a problem for therapeutic gene editing in vivo. Here, the authors compare the CRISPR-Cas9TX variant with CRISPR-Cas9 and show elimination of chromosomal translocations and reduction of AVV integration when targeting Vegfa for the treatment of age-related macular degeneration in a mouse model.
- Jianhang Yin
- , Kailun Fang
- & Jiazhi Hu
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Article
| Open AccessGene augmentation prevents retinal degeneration in a CRISPR/Cas9-based mouse model of PRPF31 retinitis pigmentosa
PRPF31-RP is a blinding disease, caused by insufficient levels of a pre-mRNA splicing factor. Here, the authors show that CRISPR-Cas9 editing of the Prpf31 gene in mice leads to retinal degeneration similar to human patients, and, in the same model, demonstrate benefits from PRPF31 gene therapy.
- Zhouhuan Xi
- , Abhishek Vats
- & Leah C. Byrne
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Article
| Open AccessEngineering antiviral immune-like systems for autonomous virus detection and inhibition in mice
The development of broad-spectrum antivirals is an important part of pandemic preparedness and response. Here the authors present ALICE, synthetic biology designer immune-like cells that act as a sense-and-destroy antiviral system can detect viruses from seven different genera, mimicking the human innate immune system.
- Yidan Wang
- , Ying Xu
- & Haifeng Ye
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Article
| Open AccessAutomated high-throughput genome editing platform with an AI learning in situ prediction model
A large number of cell disease models with pathogenic SNVs are needed. Here the authors report an automated high-throughput platform to perform the genome editing process from gRNA design to the analysis of the editing results; they characterise in situ base editing outcomes.
- Siwei Li
- , Jingjing An
- & Meng Wang
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Article
| Open AccessSelf-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo
Here, authors show that precise integration of an rAAV vector into a chromosomal target locus results in expression of a self-cleaving guide RNA. This recombination event creates genetic resistance to a hepatotoxic compound and enables in vivo expansion of gene-edited hepatocytes using a selection drug.
- Amita Tiyaboonchai
- , Anne Vonada
- & Markus Grompe
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Article
| Open AccessLow-dose AAV-CRISPR-mediated liver-specific knock-in restored hemostasis in neonatal hemophilia B mice with subtle antibody response
Here, the authors develop an AAV-CRISPR mediated somatic knock-in. They apply this system to restore hemostasis in neonatal hemophilia B mice and show liver-specificity of the knock-in and low serum antibody production.
- Xiangjun He
- , Zhenjie Zhang
- & Bo Feng
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Article
| Open AccessCompact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo
Zinc finger (ZF) arrays are programmable DNA-binding proteins. Here the authors report ZF-DddA-derived cytosine base editors (DdCBEs) and optimise their architectures to improve targeting; they apply these variants in vitro and in vivo to mitochondrial base editing and show higher editing than ZF deaminases.
- Julian C. W. Willis
- , Pedro Silva-Pinheiro
- & David R. Liu
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Article
| Open AccessCholesterol-induced LRP3 downregulation promotes cartilage degeneration in osteoarthritis by targeting Syndecan-4
This study demonstrates a role of cholesterol metabolism-related gene, Lrp3, in cartilage degeneration and osteoarthritis pathogenesis. LRP3 positively regulates cartilage extracellular matrix metabolism by targeting syndecan-4 via Ras signalling, implicating the cholesterol-LRP3-SDC4 axis in osteoarthritic cartilage degeneration.
- Chenxi Cao
- , Yuanyuan Shi
- & Yingfang Ao
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Article
| Open AccessCDKN1A is a target for phagocytosis-mediated cellular immunotherapy in acute leukemia
Inducing phagocytic capacities of tumour-associated macrophages to eliminate cancer cells is a promising immunotherapy. Here, the authors show that engineered macrophages overexpressing CDKN1A/p21 reduce leukaemic tumour burden and increase survival in preclinical mouse models of human T-ALL.
- Awatef Allouch
- , Laurent Voisin
- & Jean-Luc Perfettini
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Article
| Open AccessBase-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression
Antoniou and colleagues used base editing to generate a variety of mutations inducing γ-globin and rescue the β-hemoglobinopathy phenotype. This strategy was safe and effective in long-term repopulating hematopoietic stem/progenitor cells.
- Panagiotis Antoniou
- , Giulia Hardouin
- & Annarita Miccio
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Article
| Open AccessCross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing
Gonzalez et al. use a viral evolutionary approach to generate cross-species compatible AAV (ccAAVs) vectors. They describe a highly potent new variant, AAV.cc47, with enhanced transduction efficiency over AAV serotype 9 and show its efficacy in different mouse models, pigs and non-human primates.
- Trevor J. Gonzalez
- , Katherine E. Simon
- & Aravind Asokan
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Article
| Open AccessIn vivo self-assembled siRNA as a modality for combination therapy of ulcerative colitis
Management of ulcerative colitis can require a combination of treatments targeting different pathways. Here the authors design a therapy for ulcerative colitis based on a multitargeted genetic circuit to simultaneously target TNF-α, B7-1 and integrin α4, and show the therapy is effective in male mice with induced or spontaneous genetic colitis.
- Xinyan Zhou
- , Mengchao Yu
- & Xi Chen
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Article
| Open AccessIn vivo lentiviral vector gene therapy to cure hereditary tyrosinemia type 1 and prevent development of precancerous and cancerous lesions
Hereditary tyrosinemia type 1 (HT1) is an inborn error of metabolism caused by a deficiency in fumarylacetoacetate hydrolase (FAH). Here, the authors show in an animal model that HT1 can be treated via in vivo portal vein administration of a lentiviral vector carrying the human FAH transgene.
- Clara T. Nicolas
- , Caitlin J. VanLith
- & Joseph B. Lillegard
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Article
| Open AccessMassively targeted evaluation of therapeutic CRISPR off-targets in cells
Thorough evaluation of CRISPR RNA-guided nucleases off-targets in cells is required for advancing gene therapies. Here the authors report SURRO-seq for the simultaneous investigation of thousands of off-target sites for therapeutic RNA-guided nucleases in cells.
- Xiaoguang Pan
- , Kunli Qu
- & Yonglun Luo
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Article
| Open AccessClonal reconstruction from co-occurrence of vector integration sites accurately quantifies expanding clones in vivo
High transduction rates of viral vectors ensure good gene delivery; however multiple integration events can occur in the same cell. Here the authors use correlations between repeated measurements of integration site abundances to estimate their mutual similarity and identify clusters of co-occurring sites.
- Sebastian Wagner
- , Christoph Baldow
- & Ingmar Glauche
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Article
| Open AccessFrequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites
The identification of events occurring at target sites is critical to determine the safety of CRISPRbased DNA editing tools. Here, the authors show that LINE-1 retrotranspositions can occur frequently at canonical CRISPR/Cas9 editing sites, but are rare with prime editors and base editors.
- Jianli Tao
- , Qi Wang
- & Roberto Chiarle