Dataset Information

Multiplexed promoterless gene expression with CRISPReader.

ABSTRACT:

Background

Genes are comprised of DNA codes and contain promoters and other control elements for reading these codes. The rapid development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has made possible the construction of a novel code-reading system with low dependency on the native control elements.

Results

We develop CRISPReader, a technology for controlling promoterless gene expression in a robust fashion. We demonstrate that this tool is highly efficient in controlling transcription and translation initiation of a targeted transgene. A notable feature of CRISPReader is the ability to "read" the open reading frames of a cluster of gene without traditional regulatory elements or other cofactors. In particular, we use this strategy to construct an all-in-one AAV-CRISPR-Cas9 system by removing promoter-like elements from the expression cassette to resolve the existing AAV packaging size problem. The compact AAV-CRISPR-Cas9 is also more efficient in transactivation, DNA cleavage, and gene editing than the dual-AAV vector encoding two separate Cas9 elements, shown by targeting both reporter and endogenous genes in vitro and in vivo.

Conclusions

CRISPReader represents a novel approach for gene regulation that enables minimal gene constructs to be expressed and can be used in potential biomedical applications.

SUBMITTER: Zhan H

PROVIDER: S-EPMC6545682 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

Multiplexed promoterless gene expression with CRISPReader.

Zhan Hengji H Zhou Qun Q Gao Qunjun Q Li Jianfa J Huang Weiren W Liu Yuchen Y

Genome biology 20190603 1

<h4>Background</h4>Genes are comprised of DNA codes and contain promoters and other control elements for reading these codes. The rapid development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has made possible the construction of a novel code-reading system with low dependency on the native control elements.<h4>Results</h4>We develop CRISPReader, a technology for controlling promoterless gene expression in a robust fashion. We demonstrate that this tool is hi ...[more]

PMID: 31159834

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Project description:Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into ∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

Project description:Many inborn errors of metabolism require life-long treatments and, in severe conditions involving the liver, organ transplantation remains the only curative treatment. Non-integrative AAV-mediated gene therapy has shown efficacy in adult patients. However, treatment in pediatric or juvenile settings, or in conditions associated with hepatocyte proliferation, may result in rapid loss of episomal viral DNA and thus therapeutic efficacy. Re-administration of the therapeutic vector later in time may not be possible due to the presence of anti-AAV neutralizing antibodies. We have previously shown the permanent rescue of the neonatal lethality of a Crigler-Najjar mouse model by applying an integrative gene-therapy based approach. Here, we targeted the human coagulation factor IX (hFIX) cDNA into a hemophilia B mouse model. Two AAV8 vectors were used: a promoterless vector with two arms of homology for the albumin locus, and a vector carrying the CRISPR/SaCas9 and the sgRNA. Treatment of neonatal P2 wild-type mice resulted in supraphysiological levels of hFIX being stable 10 months after dosing. A single injection of the AAV vectors into neonatal FIX KO mice also resulted in the stable expression of above-normal levels of hFIX, reaching up to 150% of the human levels. Mice subjected to tail clip analysis showed a clotting capacity comparable to wild-type animals, thus demonstrating the rescue of the disease phenotype. Immunohistological analysis revealed clusters of hFIX-positive hepatocytes. When we tested the approach in adult FIX KO mice, we detected hFIX in plasma by ELISA and in the liver by western blot. However, the hFIX levels were not sufficient to significantly ameliorate the bleeding phenotype upon tail clip assay. Experiments conducted using a AAV donor vectors containing the eGFP or the hFIX cDNAs showed a higher recombination rate in P2 mice compared to adult animals. With this study, we demonstrate an alternative gene targeting strategy exploiting the use of the CRISPR/SaCas9 platform that can be potentially applied in the treatment of pediatric patients suffering from hemophilia, also supporting its application to other liver monogenic diseases. For the treatment of adult patients, further studies for the improvement of targeting efficiency are still required.

Dataset Information

Multiplexed promoterless gene expression with CRISPReader.

Background

Results

Conclusions

Publications

Multiplexed promoterless gene expression with CRISPReader.

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