CRISPR/Cas9 – Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

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HPRT As a Selectable Safe Harbor for Transgenesis

Blood ◽

10.1182/blood.v124.21.4796.4796 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4796-4796

Author(s):

Anthony Conway ◽

Josée Laganière ◽

David E Paschon ◽

Katrin Hacke ◽

Noriyuki Kasahara ◽

...

Keyword(s):

Fold Increase ◽

Selective Growth ◽

Live Cells ◽

Zinc Finger Nucleases ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Targeted Integration ◽

Selection Of

Abstract A current limitation in gene therapy is obtaining a sufficient number of modified cells to produce a therapeutic effect in vivo. In several diseases, correction of a mutant allele confers a selective growth advantage to the modified cells, thus enhancing efficacy with moderate initial modification. For most diseases, however, there is no selective advantage to the corrected cells. One potential strategy to address this limitation is in vivo selection of modified cells using pharmacological agents. It has previously been shown that 6-thioguanine (6-TG), an FDA-approved chemotherapeutic small molecule, is cytotoxic to cells expressing the enzyme HPRT, allowing for selective growth of HPRT knockout cells. Knockout of HPRT can be achieved by creating a nonsense mutation in an upstream exon, or by terminating splicing by introducing a large transgene into an intron. To allow for selectable transgenesis of only cells which have undergone targeted integration (TI), engineered zinc-finger nucleases (ZFNs) were used to insert a virally-delivered transgene into an HPRT intron. After two weeks of in vitro 6-TG selection following genome modification, a 95-fold increase in TI was observed in pooled K562 cell populations to a final level of 72% TI, whereas a 30-fold increase in transgene-expressing live cells was seen in peripheral blood-mobilized primary CD34+ cells resulting in 90% transgene-positive live cells. Furthermore, a 72-fold increase in transgene mRNA transcript was observed after two weeks of erythroid differentiation and 6-TG selection of CD34+ cells compared to unselected genome-modified controls. These results represent an important step in developing hematopoietic stem cell (HSC)-based gene therapies, as well as a platform technology for creating gene-modified HSC populations with high proportions of therapeutic transgene expression via precise, targeted integration of a transgene of interest. Disclosures Conway: Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment. Cost:Sangamo Biosciences: Employment.

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710. Stem Cell Gene Therapy: Synergizing Improved Gene Repair/Modification & Targeted Integration of Transgenes to Transient In Vivo Topical Minigene Expression

Molecular Therapy ◽

10.1016/j.ymthe.2004.06.617 ◽

2004 ◽

Vol 9 ◽

pp. S271

Keyword(s):

Gene Therapy ◽

Stem Cell ◽

Gene Repair ◽

Targeted Integration ◽

Cell Gene ◽

Stem Cell Gene ◽

Stem Cell Gene Therapy ◽

Minigene Expression

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311. In Vivo Zinc Finger Nuclease-Mediated Targeted Integration of a Glucose-6-Phosphatase Transgene Enhances Biochemical Correction in Mice with Glycogen Storage Disease Type IA

Molecular Therapy ◽

10.1016/s1525-0016(16)33120-3 ◽

2016 ◽

Vol 24 ◽

pp. S125

Author(s):

Dustin J. Landau ◽

Elizabeth D. Brooks ◽

Pablo Pablo Perez-Pinera ◽

Hiruni Amarasekara ◽

Adam Mefferd ◽

...

Keyword(s):

Zinc Finger ◽

Glycogen Storage Disease ◽

Storage Disease ◽

Glycogen Storage Disease Type ◽

Glycogen Storage ◽

Disease Type ◽

Zinc Finger Nuclease ◽

Targeted Integration ◽

Type Ia

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A Safe Harbor-Targeted CRISPR/Cas9 Homology Independent Targeted Integration (HITI) System for Multi-Modality Reporter Gene-Based Cell Tracking

10.1101/2020.02.10.942672 ◽

2020 ◽

Cited By ~ 3

Author(s):

John J Kelly ◽

Moe Saee-Marand ◽

Nivin N Nyström ◽

Yuanxin Chen ◽

Melissa M Evans ◽

...

Keyword(s):

Reporter Gene ◽

Cell Tracking ◽

Integration Site ◽

Reporter Genes ◽

Safe Harbor ◽

Enhanced Mri ◽

Contrast Enhanced ◽

Targeted Integration ◽

Contrast Enhanced Mri

AbstractImaging reporter genes can provide valuable, longitudinal information on the biodistribution, growth and survival of engineered cells in preclinical models and patients. A translational bottleneck to using reporter genes in patients is the necessity to engineer cells with randomly-integrating vectors. CRISPR/Cas9 targeted knock-in of reporter genes at a genomic safe harbor locus such as adeno-associated virus integration site 1 (AAVS1) may overcome these safety concerns. Here, we built Homology Independent Targeted Integration (HITI) CRISPR/Cas9 minicircle donors for precise AAVS1-targeted simultaneous knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency at the AAVS1 site using HITI vectors compared to homology-directed repair donor vectors. Characterization of select HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significantly increased Oatp1a1-mediated uptake of the clinically-approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. As few as 106 Oatp1a1-expressing cells in a 50 µl subcutaneous injection could be detected in vivo with contrast-enhanced MRI. Contrast-enhanced MRI also improved the conspicuity of both sub-cutaneous and metastatic Oatp1a1-expressing tumours prior to them being palpable or even readily visible on pre-contrast images. Our work demonstrates the first CRISPR/Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multi-modal longitudinal in vivo imaging of cells. This work lays the foundation for safer, non-viral reporter gene tracking of multiple cell types.

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GeneWeld: a method for efficient targeted integration directed by short homology

10.1101/431627 ◽

2018 ◽

Cited By ~ 5

Author(s):

Wesley A. Wierson ◽

Jordan M. Welker ◽

Maira P. Almeida ◽

Carla M. Mann ◽

Dennis A. Webster ◽

...

Keyword(s):

Genome Engineering ◽

High Efficiency ◽

High Specificity ◽

Strand Break ◽

Double Strand Break ◽

Genomic Locus ◽

Multiple Loci ◽

Integration Strategy ◽

Targeted Integration

AbstractChoices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids forGeneTagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates thatin vivotargeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand breakin vivo. Our results suggest that the length of homology is not important in the design of knock-in vectors but rather how the homology is presented to a double strand break in the genome. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR and TALEN compatible systems.

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