Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

Karnan, Sivasundaram; Ota, Akinobu; Konishi, Yuko; Wahiduzzaman, Md; Hosokawa, Yoshitaka; Konishi, Hiroyuki

doi:10.1093/nar/gkv1338

Cited by 16 publications

(12 citation statements)

References 53 publications

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“…Promoter trap encodes for a promoterless GFP reporter (with bovine growth hormone polyA) followed by a puromycin resistance gene under the control of the human phosphoglycerate kinase 1 (PGK) promoter with a SV40 polyA. For intron traps, we added a synthetic intron with splice acceptor site (adapted from 62 and a self-cleaving peptide from porcine teschovirus-1 (P2A) 63 in frame (+0 or +2) at 5′ of GFP cDNA ( Supplementary Fig. 2a and Supplementary Methods).…”

Section: Methodsmentioning

confidence: 99%

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

et al. 2020

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Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.

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Section: Methodsmentioning

confidence: 99%

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

et al. 2020

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“…Thus, for in vivo applications, it would likely be beneficial to minimize the duration of Cas9 mediated genome editing to the time necessary for Cas9 to edit its intended target site and this has been, in part, the motivation for developing previously described inducible CRISPR/Cas9 genome editing systems (Davis et al, 2015 ; Nihongaki et al, 2015 ; Zetsche et al, 2015 ). However, while some of CRISPR/Cas9 systems have been adapted for AAV delivery (Ran et al, 2015 ; Swiech et al, 2015 ; Karnan et al, 2016 ; Yang et al, 2016 ), none of these AAV based systems, to date, have been adapted to allow the genome editing function to be temporally regulated.…”

Section: Introductionmentioning

confidence: 99%

The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

Solı́s

Holehonnur

et al. 2016

Front. Mol. Neurosci.

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The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well.

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“…We therefore conclude that the use of 2A peptide‐based vectors is preferable for surely isolating homologous recombinants particularly when the target gene expression is low, but IRES‐based vectors have a potential to confer higher targeting efficiencies at ‘easy‐to‐target’ loci, such as HPRT . With regard to the advantage of employing a 2A peptide sequence, Konishi and colleagues have recently reached essentially the same conclusion using various types of rAAV‐based targeting vectors . In mouse ES cells, several low expression genes were not targeted with IRES‐based exon‐trap vectors .…”

Section: Discussionmentioning

confidence: 87%

Mechanistic basis for increased human gene targeting by promoterless vectors—roles of homology arms and Rad54 paralogs

2017

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Gene targeting by homologous recombination provides the definitive tool for analyzing gene function. Promoterless vectors, which do not possess a promoter to drive marker gene expression, confer higher targeting efficiencies than conventional vectors due to the reduced number of drug-resistant clones. We here show that gene-targeting efficiency is typically ≥ 25% with the use of exon-trapping-type promoterless vectors in a human diploid cell line, Nalm-6. The efficiency of exon-trapping gene targeting was correlated with the level of target gene expression when a 2A peptide sequence was linked to the marker gene. Intriguingly, total arm length was not necessarily a determinant of targeting efficiency, as longer arms tend to enhance both homologous (targeted) and nonhomologous (nontargeted) integration of the vector; rather, the presence of an exon in the 5' arm led to a decreased targeting efficiency. Strikingly, loss of Rad54 did not severely affect the targeting efficiency of exon-trap vectors. Moreover, additional deletion of the Rad54 paralog Rad54B had limited impact on the high-efficiency gene targeting. These results indicate that targeted integration occurs in human cells even when both Rad54 and Rad54B are missing. These studies provide additional important insight into the contribution of various DNA repair factors on the targeting mechanics.

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Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

Cited by 16 publications

References 53 publications

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

Mechanistic basis for increased human gene targeting by promoterless vectors—roles of homology arms and Rad54 paralogs

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