scholarly journals Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break

2019 ◽  
Author(s):  
Geoffrey L. Rogers ◽  
Hsu-Yu Chen ◽  
Heidy Morales ◽  
Paula M. Cannon
Keyword(s):  
Homologous Recombination ◽  
Progenitor Cells ◽  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Dna Breaks ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem ◽  
Double Stranded Dna ◽  
Dna Break

AbstractAdeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem/progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 or AAVS1 loci in human HSPCs and cell lines. As a control we used AAV6, which effectively edits HSPCs, but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results therefore do not support that clade F AAVs can perform high efficiency genome editing in the absence of a DSB.

scholarly journals Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

2018 ◽  
Vol 115 (31) ◽  
pp. E7379-E7388 ◽  
Author(s):  
Laura J. Smith ◽  
Jason Wright ◽  
Gabriella Clark ◽  
Taihra Ul-Hasan ◽  
Xiangyang Jin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Homologous Recombination ◽  
Genome Editing ◽  
High Efficiency ◽  
Dna Breaks ◽  
Hematopoietic Stem ◽  
A Genome ◽  
Component Approach ◽  
Il2rg Gene

The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34+cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases.

scholarly journals High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells By AAV6 Vectors: strategies for Overcoming Donor-Variation and Implications in Genome Editing

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5889-5889
Author(s):  
Chen Ling ◽  
Kanit Bhukhai ◽  
Zifei Yin ◽  
Mengqun Tan ◽  
Mervin Yoder ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Genome Editing ◽  
High Efficiency ◽  
Ex Vivo ◽  
Transduction Efficiency ◽  
Triple Mutant ◽  
Hematopoietic Stem ◽  
Combined Use ◽  
Donor Variation ◽  
Viral Capsid Proteins

Abstract We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705+Y731F+T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, 'cross-transduction'. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. Disclosures Leboulch: bluebird bio: Patents & Royalties. Payen:bluebird bio: Patents & Royalties. Srivastava:AGTC; Genzyme: Patents & Royalties.

scholarly journals High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Chen Ling ◽  
Kanit Bhukhai ◽  
Zifei Yin ◽  
Mengqun Tan ◽  
Mervin C. Yoder ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Genome Editing ◽  
High Efficiency ◽  
Hematopoietic Stem ◽  
Donor Variation

scholarly journals 10. High-Efficiency Transduction of Primary Human CD34+ Hematopoietic Stem/Progenitor Cells by AAV6 Serotype Vectors: Strategies for Overcoming Donor Variation and Implications in Genome Editing

2016 ◽  
Vol 24 ◽  
pp. S5-S6
Author(s):  
Zifei Yin ◽  
Kanit Bukhai ◽  
George Aslanidi ◽  
Chen Ling ◽  
Mengqun Tan ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Genome Editing ◽  
High Efficiency ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Donor Variation

scholarly journals DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Prasun Chakraborty ◽  
Kevin Hiom
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair ◽  
Dna End Resection ◽  
End Resection ◽  
Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells

2016 ◽  
Vol 36 (1) ◽  
Author(s):  
Megan D. Hoban ◽  
Zulema Romero ◽  
Gregory J. Cost ◽  
Matthew Mendel ◽  
Michael Holmes ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Genome Editing ◽  
Hematopoietic Stem

Excessive Hematopoietic Stem Cell Proliferation Leads to Chromosomal Instability

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4772-4772
Author(s):  
Liliana Souza ◽  
Natalyn Hawk ◽  
Sweta Sengupta ◽  
Carlos Cabrera ◽  
Morgan L. McLemore
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Chromosomal Instability ◽  
Wild Type ◽  
Dna Breaks ◽  
Hematopoietic Stem ◽  
Stem Cell Proliferation ◽  
Double Stranded Dna

Abstract Truncation mutations in the granulocyte colony stimulating factor receptor (G-CSFR), common in severe congenital neutropenia (SCN), lead to excessive stem cell proliferation in response to G-CSF. These G-CSFR mutants are (at least indirectly) implicated in the progression of these patients to acute leukemia. Since SCN patients require continuous G-CSF treatment throughout their lifespan, we hypothesize that excessive stem cell proliferation can lead to DNA damage. Stem cells are relatively quiescent and rarely enter the cell cycle under normal conditions. During the cell cycle cells generate approximately 5000 single strand DNA lesions per nucleus (Vilenchik and Knudson, 2003). Approximately 1% of these lesions are ultimately converted to double strand DNA breaks (DSBs). Hematopoietic stem cells are found within the Sca+ ckit+ Lin- (KLS) population. Wild type and mice bearing a mutant G-CSFR similar to that found in patients with SCN were treated with G-CSF. After 21 days of treatment with G-CSF (10 ug/kg/day), the KLS population in the bone marrow increased four-fold in wild type mice and eight-fold in mutant mice. We isolated Lin-Sca+ bone marrow cells from these G-CSF treated mice and evaluated for the presence of double stranded DNA breaks by staining with anti-phospho-H2AX by immunofluorescence. H2AX is a histone whose phosphorylated form localizes to the site of double stranded DNA breaks. The results showed that there is an 8-fold increase in the DSB in wild type Lin-Sca+ and 10-fold in mutant Lin-Sca+ when compared to cells from untreated mice. This data suggests that excessive proliferation can contribute to an increase in DSBs in hematopoietic stem cells. Investigation of potential mechanisms contributing to DSB formation are ongoing. Understanding the causes and trends of chromosomal instability would improve our understanding of leukemogenesis and potentially reveal novel treatment strategies.

scholarly journals Genomic Editing of the HIV-1 Coreceptor CCR5 in Adult Hematopoietic Stem and Progenitor Cells Using Zinc Finger Nucleases

2013 ◽  
Vol 21 (6) ◽  
pp. 1259-1269 ◽  
Author(s):  
Lijing Li ◽  
Ludmila Krymskaya ◽  
Jianbin Wang ◽  
Jill Henley ◽  
Anitha Rao ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Zinc Finger ◽  
Zinc Finger Nucleases ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Genomic Editing ◽  
Hiv 1

Adipose tissue is an extramedullary reservoir for functional hematopoietic stem and progenitor cells

Blood ◽  
2010 ◽  
Vol 115 (5) ◽  
pp. 957-964 ◽  
Author(s):  
Jinah Han ◽  
Young Jun Koh ◽  
Hye Rin Moon ◽  
Hyun Gee Ryoo ◽  
Chung-Hyun Cho ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Progenitor Cells ◽  
High Efficiency ◽  
Stromal Vascular Fraction ◽  
Hematopoietic Stem ◽  
Blood Transplantation ◽  
Variable Extent ◽  
Stem And Progenitor Cells

Abstract The stromal vascular fraction (SVF) in adipose tissue contains a pool of various stem and progenitor cells, but the existence of hematopoietic stem and progenitor cells (HSPCs) in the SVF has not been seriously considered. We detected the presence of HSPCs in the SVF by phenotypically probing with Lin−Sca-1+c-kit+ (LSK) and functionally confirming the presence using colony-forming cell assay and assessing the long-term multilineage reconstitution ability after SVF transplantation. The LSK population in the SVF was 0.004% plus or minus 0.001%, and 5 × 105 freshly isolated SVF cells gave rise to 13 plus or minus 4 multilineage colonies. In addition, 0.15% plus or minus 0.03% of SVF cells was home to bone marrow (BM), especially near vascular and endosteal regions, 24 hours after blood transplantation. SVF transplantation was capable of generating a long-term (> 16 weeks), but variable extent (2.1%-32.1%) multilineage reconstitution in primary recipients, which was subsequently transferred to the secondary recipients by BM transplantation. All HSPCs within the SVF originated from the BM. Furthermore, the granulocyte–colony-stimulating factor (G-CSF) mobilization of HSPCs from BM markedly elevated the number of phenotypic and functional HSPCs in the SVF, which induced a high efficiency long-term reconstitution in multilineage hematopoiesis in vivo. Our results provide compelling evidence that adipose tissue is a novel extramedullary tissue possessing phenotypic and functional HSPCs.

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