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Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders. ​

Literature Information ​

DOI10.1038/s41598-017-16279-4
PMID29170458
JournalScientific reports
Impact Factor3.9
JCR QuartileQ1
Publication Year2017
Times Cited47
KeywordsCRISPR gene editing, allele-specific, autosomal dominant disorders
Literature TypeJournal Article, Research Support, Non-U.S. Gov't
ISSN2045-2322
Pages16174
Issue7(1)
AuthorsKathleen A Christie, David G Courtney, Larry A DeDionisio, Connie Chao Shern, Shyamasree De Majumdar, Laura C Mairs, M Andrew Nesbit, C B Tara Moore

TL;DR ​

This study explores the use of CRISPR/Cas9 for treating autosomal dominant genetic disorders, specifically through allele-specific gene disruption via non-homologous end-joining (NHEJ) in corneal epithelial cells. The findings reveal that while cleavage using a SNP-derived PAM provides stringent allele specificity, the guide-specific approach fails to differentiate between alleles, underscoring the importance of careful guide design to minimize off-target effects and enhance therapeutic potential.

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CRISPR gene editing · allele-specific · autosomal dominant disorders

Abstract ​

CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at "off-target" sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.

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Primary Questions Addressed ​

  1. What are the potential implications of using SNP-derived PAMs in CRISPR gene editing for other genetic disorders beyond TGFBI corneal dystrophies?
  2. How might the findings regarding guide-specific approaches inform future research on CRISPR design and optimization for allele-specific editing?
  3. In what ways can the long-term effects of CRISPR-induced gene disruption be evaluated in vivo, particularly in the context of autosomal dominant diseases?
  4. What alternative methods exist for enhancing the specificity of CRISPR/Cas9 systems to minimize off-target effects during allele-specific gene editing?
  5. How can the principles of allele-specific gene disruption be applied to other therapeutic areas, such as cancer or rare genetic disorders?

Key Findings ​

Research Background and Objective ​

Autosomal dominant disorders are genetic conditions caused by mutations in a single allele, often leading to significant clinical manifestations. Current gene editing technologies, such as CRISPR, lack the precision needed to target specific alleles without affecting the wild-type allele. This study aims to develop a personalized, allele-specific CRISPR gene editing strategy to treat these disorders, particularly focusing on TGFBI mutations associated with corneal dystrophies.

Main Methods/Materials/Experimental Design ​

The study employs a multi-step approach to design and test allele-specific CRISPR/Cas9 constructs targeting TGFBI mutations. The workflow includes the following key steps:

Mermaid diagram
  1. Identification of Mutations: The study first identifies specific TGFBI mutations that can be targeted for editing.
  2. sgRNA Design: Specific guide RNAs (sgRNAs) are designed for each mutation, ensuring they can selectively bind to the mutant allele.
  3. Cloning: The sgRNAs are cloned into CRISPR/Cas9 constructs for expression in target cells.
  4. Transfection: These constructs are then transfected into cells harboring the TGFBI mutations.
  5. Evaluation: The editing efficiency and specificity of the CRISPR system are assessed, along with potential off-target effects.
  6. Confirmation: Successful editing is confirmed through sequencing.

Key Results and Findings ​

  • The allele-specific CRISPR constructs demonstrated high editing efficiency for the targeted TGFBI mutations while maintaining low off-target activity.
  • Specificity was confirmed through a series of assays that showed minimal effects on the wild-type allele.
  • The study identified several novel PAM (protospacer adjacent motif) sequences generated by the mutations, which facilitated the design of effective sgRNAs.

Main Conclusions/Significance/Innovation ​

The research presents a novel approach to allele-specific gene editing, which could significantly enhance the treatment of autosomal dominant disorders. By utilizing a personalized CRISPR strategy, the study demonstrates that it is possible to selectively target mutant alleles while sparing the wild-type, thus minimizing potential adverse effects associated with broader gene editing strategies. This work lays the groundwork for future clinical applications of personalized gene therapies.

Research Limitations and Future Directions ​

  • Limitations: The study primarily focuses on in vitro models, and further research is needed to evaluate the efficacy and safety of this approach in vivo. Additionally, the potential for off-target effects, although minimized, still requires thorough investigation.
  • Future Directions: Future studies should explore the application of this technique in animal models and eventually in human clinical trials. Expanding the approach to other genetic disorders and refining the specificity of the CRISPR system will also be essential for broader therapeutic applications.

References ​

  1. CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting. - D G Courtney;J E Moore;S D Atkinson;E Maurizi;E H A Allen;D M L Pedrioli;W H I McLean;M A Nesbit;C B T Moore - Gene therapy (2016)
  2. Clinical findings and treatments of granular corneal dystrophy type 2 (avellino corneal dystrophy): a review of the literature. - Kyung Eun Han;Tae-im Kim;Woo Suk Chung;Seung-il Choi;Bong-yoon Kim;Eung Kweon Kim - Eye & contact lens (2010)
  3. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. - Wenyan Jiang;David Bikard;David Cox;Feng Zhang;Luciano A Marraffini - Nature biotechnology (2013)
  4. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. - Xuebing Wu;David A Scott;Andrea J Kriz;Anthony C Chiu;Patrick D Hsu;Daniel B Dadon;Albert W Cheng;Alexandro E Trevino;Silvana Konermann;Sidi Chen;Rudolf Jaenisch;Feng Zhang;Phillip A Sharp - Nature biotechnology (2014)
  5. Development of allele-specific gene-silencing siRNAs for TGFBI Arg124Cys in lattice corneal dystrophy type I. - David G Courtney;Sarah D Atkinson;Johnny E Moore;Eleonora Maurizi;Chiara Serafini;Graziella Pellegrini;Graeme C Black;Forbes D Manson;Gary H F Yam;Caroline J Macewen;Edwin H A Allen;W H Irwin McLean;C B Tara Moore - Investigative ophthalmology & visual science (2014)
  6. Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency. - Jian-Ping Zhang;Xiao-Lan Li;Amanda Neises;Wanqiu Chen;Lin-Ping Hu;Guang-Zhen Ji;Jun-Yao Yu;Jing Xu;Wei-Ping Yuan;Tao Cheng;Xiao-Bing Zhang - Scientific reports (2016)
  7. siRNA silencing of the mutant keratin 12 allele in corneal limbal epithelial cells grown from patients with Meesmann's epithelial corneal dystrophy. - David G Courtney;Sarah D Atkinson;Edwin H A Allen;Johnny E Moore;Colum P Walsh;Deena M Leslie Pedrioli;Caroline J MacEwen;Graziella Pellegrini;Eleonora Maurizi;Chiara Serafini;Monica Fantacci;Haihui Liao;Alan D Irvine;W H Irwin McLean;C B Tara Moore - Investigative ophthalmology & visual science (2014)
  8. Pathogenesis and treatments of TGFBI corneal dystrophies. - Kyung Eun Han;Seung-il Choi;Tae-im Kim;Yong-sun Maeng;R Doyle Stulting;Yong Woo Ji;Eung Kweon Kim - Progress in retinal and eye research (2016)
  9. Genome engineering using the CRISPR-Cas9 system. - F Ann Ran;Patrick D Hsu;Jason Wright;Vineeta Agarwala;David A Scott;Feng Zhang - Nature protocols (2013)
  10. In vivo genome editing using Staphylococcus aureus Cas9. - F Ann Ran;Le Cong;Winston X Yan;David A Scott;Jonathan S Gootenberg;Andrea J Kriz;Bernd Zetsche;Ophir Shalem;Xuebing Wu;Kira S Makarova;Eugene V Koonin;Phillip A Sharp;Feng Zhang - Nature (2015)

Literatures Citing This Work ​

  1. Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity. - Christopher R Cromwell;Keewon Sung;Jinho Park;Amanda R Krysler;Juan Jovel;Seong Keun Kim;Basil P Hubbard - Nature communications (2018)
  2. Gene therapy and genome surgery in the retina. - James E DiCarlo;Vinit B Mahajan;Stephen H Tsang - The Journal of clinical investigation (2018)
  3. Gene editing in the context of an increasingly complex genome. - K Blighe;L DeDionisio;K A Christie;B Chawes;S Shareef;T Kakouli-Duarte;C Chao-Shern;V Harding;R S Kelly;L Castellano;J Stebbing;J A Lasky-Su;M A Nesbit;C B T Moore - BMC genomics (2018)
  4. Regulatory SNPs and their widespread effects on the transcriptome. - Vasily M Merkulov;Elena Yu Leberfarb;Tatiana I Merkulova - Journal of biosciences (2018)
  5. Synthesis and Evaluation of pH-Sensitive Multifunctional Lipids for Efficient Delivery of CRISPR/Cas9 in Gene Editing. - Da Sun;Zhanhu Sun;Hongfa Jiang;Amita M Vaidya;Rui Xin;Nadia R Ayat;Andrew L Schilb;Peter L Qiao;Zheng Han;Amirreza Naderi;Zheng-Rong Lu - Bioconjugate chemistry (2019)
  6. Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs. - Sachiko Okamoto;Yasunori Amaishi;Izumi Maki;Tatsuji Enoki;Junichi Mineno - Scientific reports (2019)
  7. Fuchs endothelial corneal dystrophy and corneal endothelial diseases: East meets West. - Y Q Soh;Viridiana Kocaba;Mauricio Pinto;Jodhbir S Mehta - Eye (London, England) (2020)
  8. AlleleAnalyzer: a tool for personalized and allele-specific sgRNA design. - Kathleen C Keough;Svetlana Lyalina;Michael P Olvera;Sean Whalen;Bruce R Conklin;Katherine S Pollard - Genome biology (2019)
  9. Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications. - Emily M Mills;Victoria L Barlow;Louis Y P Luk;Yu-Hsuan Tsai - Cell biology and toxicology (2020)
  10. Normal peripheral blood neutrophil numbers accompanying ELANE whole gene deletion mutation. - Marshall S Horwitz;Mercy Y Laurino;Siobán B Keel - Blood advances (2019)

... (37 more literatures)

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