skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity

Abstract

The retargeting of protein–DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date. Specificity is derived from the combined contributions of DNA-contacting residues and of neighboring residues that influence local structural organization. Changes in specificity are facilitated by the ability of all those residues to readily exchange both form and function. The fidelity of recognition is not precisely correlated with the fraction or total number of residues in the protein–DNA interface that are actually involved in DNA contacts, including directional hydrogen bonds. The plasticity of the DNA-recognition surface of this protein, which allows substantial retargeting of recognition specificity without requiring significant alteration of the surrounding protein architecture, reflects the ability of the corresponding genetic elements to maintain mobility and persistence in the face of genetic driftmore » within potential host target sites.« less

Authors:
 [1];  [1];  [1];  [2];  [2];  [2];  [3];  [3];  [3];  [3]; ORCiD logo [1]
  1. Basic Sciences Division, Fred Hutchinson Cancer Research Center,Seattle, WA (United States)
  2. Bluebird Bio Inc., Seattle, WA (United States)
  3. Imperial College of London, Department of Life Sciences, South Kensington Campus, London (United Kingdom)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS); Bill and Melinda Gates Foundation; Fred Hutchinson Cancer Center; Bluebird Bio, Inc.
OSTI Identifier:
1625567
Grant/Contract Number:  
AC02-05CH11231; R01 GM105691
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 45; Journal Issue: 14; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
Biochemistry & Molecular Biology

Citation Formats

Werther, Rachel, Hallinan, Jazmine P., Lambert, Abigail R., Havens, Kyle, Pogson, Mark, Jarjour, Jordan, Galizi, Roberto, Windbichler, Nikolai, Crisanti, Andrea, Nolan, Tony, and Stoddard, Barry L. Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity. United States: N. p., 2017. Web. doi:10.1093/nar/gkx544.
Werther, Rachel, Hallinan, Jazmine P., Lambert, Abigail R., Havens, Kyle, Pogson, Mark, Jarjour, Jordan, Galizi, Roberto, Windbichler, Nikolai, Crisanti, Andrea, Nolan, Tony, & Stoddard, Barry L. Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity. United States. doi:10.1093/nar/gkx544.
Werther, Rachel, Hallinan, Jazmine P., Lambert, Abigail R., Havens, Kyle, Pogson, Mark, Jarjour, Jordan, Galizi, Roberto, Windbichler, Nikolai, Crisanti, Andrea, Nolan, Tony, and Stoddard, Barry L. Tue . "Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity". United States. doi:10.1093/nar/gkx544. https://www.osti.gov/servlets/purl/1625567.
@article{osti_1625567,
title = {Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity},
author = {Werther, Rachel and Hallinan, Jazmine P. and Lambert, Abigail R. and Havens, Kyle and Pogson, Mark and Jarjour, Jordan and Galizi, Roberto and Windbichler, Nikolai and Crisanti, Andrea and Nolan, Tony and Stoddard, Barry L.},
abstractNote = {The retargeting of protein–DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date. Specificity is derived from the combined contributions of DNA-contacting residues and of neighboring residues that influence local structural organization. Changes in specificity are facilitated by the ability of all those residues to readily exchange both form and function. The fidelity of recognition is not precisely correlated with the fraction or total number of residues in the protein–DNA interface that are actually involved in DNA contacts, including directional hydrogen bonds. The plasticity of the DNA-recognition surface of this protein, which allows substantial retargeting of recognition specificity without requiring significant alteration of the surrounding protein architecture, reflects the ability of the corresponding genetic elements to maintain mobility and persistence in the face of genetic drift within potential host target sites.},
doi = {10.1093/nar/gkx544},
journal = {Nucleic Acids Research},
number = 14,
volume = 45,
place = {United States},
year = {2017},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

Functional Specificity of a Hox Protein Mediated by the Recognition of Minor Groove Structure
journal, November 2007


The role of DNA shape in protein–DNA recognition
journal, October 2009

  • Rohs, Remo; West, Sean M.; Sosinsky, Alona
  • Nature, Vol. 461, Issue 7268
  • DOI: 10.1038/nature08473

Probing DNA shape and methylation state on a genomic scale with DNase I
journal, April 2013

  • Lazarovici, A.; Zhou, T.; Shafer, A.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 16
  • DOI: 10.1073/pnas.1216822110

Diversity in DNA recognition by p53 revealed by crystal structures with Hoogsteen base pairs
journal, April 2010

  • Kitayner, Malka; Rozenberg, Haim; Rohs, Remo
  • Nature Structural & Molecular Biology, Vol. 17, Issue 4
  • DOI: 10.1038/nsmb.1800

Absence of a simple code: how transcription factors read the genome
journal, September 2014


Coevolution of a Homing Endonuclease and Its Host Target Sequence
journal, October 2007

  • Scalley-Kim, Michelle; McConnell-Smith, Audrey; Stoddard, Barry L.
  • Journal of Molecular Biology, Vol. 372, Issue 5
  • DOI: 10.1016/j.jmb.2007.07.052

Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity
journal, June 2016


Exploitation of binding energy for catalysis and design
journal, October 2009

  • Thyme, Summer B.; Jarjour, Jordan; Takeuchi, Ryo
  • Nature, Vol. 461, Issue 7268
  • DOI: 10.1038/nature08508

Computational redesign of endonuclease DNA binding and cleavage specificity
journal, June 2006

  • Ashworth, Justin; Havranek, James J.; Duarte, Carlos M.
  • Nature, Vol. 441, Issue 7093
  • DOI: 10.1038/nature04818

Mutations altering the cleavage specificity of a homing endonuclease
journal, September 2002


A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences
journal, November 2006

  • Smith, Julianne; Grizot, Sylvestre; Arnould, Sylvain
  • Nucleic Acids Research, Vol. 34, Issue 22
  • DOI: 10.1093/nar/gkl720

Heritable targeted mutagenesis in maize using a designed endonuclease
journal, January 2010


Engineered I-CreI Derivatives Cleaving Sequences from the Human XPC Gene can Induce Highly Efficient Gene Correction in Mammalian Cells
journal, August 2007

  • Arnould, Sylvain; Perez, Christophe; Cabaniols, Jean-Pierre
  • Journal of Molecular Biology, Vol. 371, Issue 1
  • DOI: 10.1016/j.jmb.2007.04.079

Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases
journal, November 2008

  • Redondo, Pilar; Prieto, Jesús; Muñoz, Inés G.
  • Nature, Vol. 456, Issue 7218
  • DOI: 10.1038/nature07343

Targeted Gene Therapy of Xeroderma Pigmentosum Cells Using Meganuclease and TALEN™
journal, November 2013


Meganuclease-Driven Targeted Integration in CHO-K1 Cells for the Fast Generation of HTS-Compatible Cell-Based Assays
journal, July 2010

  • Cabaniols, Jean-Pierre; Ouvry, Christine; Lamamy, Véronique
  • Journal of Biomolecular Screening, Vol. 15, Issue 8
  • DOI: 10.1177/1087057110375115

Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene ( MS26 ) using a re-designed I- Cre I homing endonuclease
journal, November 2013

  • Djukanovic, Vesna; Smith, Jeff; Lowe, Keith
  • The Plant Journal, Vol. 76, Issue 5
  • DOI: 10.1111/tpj.12335

Targeted DNA excision in Arabidopsis by a re-engineered homing endonuclease
journal, January 2012

  • Antunes, Mauricio S.; Smith, J. Jeff; Jantz, Derek
  • BMC Biotechnology, Vol. 12, Issue 1
  • DOI: 10.1186/1472-6750-12-86

Targeted molecular trait stacking in cotton through targeted double‐strand break induction
journal, June 2013

  • D'Halluin, Kathleen; Vanderstraeten, Chantal; Hulle, Jolien
  • Plant Biotechnology Journal, Vol. 11, Issue 8
  • DOI: 10.1111/pbi.12085

Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease
journal, July 2009

  • Grizot, Sylvestre; Smith, Julianne; Daboussi, Fayza
  • Nucleic Acids Research, Vol. 37, Issue 16
  • DOI: 10.1093/nar/gkp548

Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus
journal, September 2010

  • Muñoz, Inés G.; Prieto, Jesús; Subramanian, Sunita
  • Nucleic Acids Research, Vol. 39, Issue 2
  • DOI: 10.1093/nar/gkq801

Generation of Rag1 ‐knockout immunodeficient rats and mice using engineered meganucleases
journal, October 2012

  • Ménoret, Séverine; Fontanière, Sandra; Jantz, Derek
  • The FASEB Journal, Vol. 27, Issue 2
  • DOI: 10.1096/fj.12-219907

Meganuclease-mediated Inhibition of HSV1 Infection in Cultured Cells
journal, April 2011

  • Grosse, Stéphanie; Huot, Nicolas; Mahiet, Charlotte
  • Molecular Therapy, Vol. 19, Issue 4
  • DOI: 10.1038/mt.2010.302

Gene Correction of a Duchenne Muscular Dystrophy Mutation by Meganuclease-Enhanced Exon Knock-In
journal, July 2013

  • Popplewell, Linda; Koo, Taeyoung; Leclerc, Xavier
  • Human Gene Therapy, Vol. 24, Issue 7
  • DOI: 10.1089/hum.2013.081

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display
journal, September 2009

  • Jarjour, Jordan; West-Foyle, Hoku; Certo, Michael T.
  • Nucleic Acids Research, Vol. 37, Issue 20
  • DOI: 10.1093/nar/gkp726

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes
journal, April 2003


Redesign of extensive protein-DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization
journal, March 2014

  • Takeuchi, R.; Choi, M.; Stoddard, B. L.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 11
  • DOI: 10.1073/pnas.1321030111

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance
journal, January 2016

  • Romano Ibarra, Guillermo S.; Paul, Biswajit; Sather, Blythe D.
  • Molecular Therapy - Nucleic Acids, Vol. 5
  • DOI: 10.1038/mtna.2016.56

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template
journal, September 2015

  • Sather, Blythe D.; Romano Ibarra, Guillermo S.; Sommer, Karen
  • Science Translational Medicine, Vol. 7, Issue 307
  • DOI: 10.1126/scitranslmed.aac5530

Reprogramming homing endonuclease specificity through computational design and directed evolution
journal, November 2013

  • Thyme, Summer B.; Boissel, Sandrine J. S.; Arshiya Quadri, S.
  • Nucleic Acids Research, Vol. 42, Issue 4
  • DOI: 10.1093/nar/gkt1212

enoLOGOS: a versatile web tool for energy normalized sequence logos
journal, July 2005

  • Workman, C. T.; Yin, Y.; Corcoran, D. L.
  • Nucleic Acids Research, Vol. 33, Issue Web Server
  • DOI: 10.1093/nar/gki439

The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive
journal, September 2013


Phaser crystallographic software
journal, July 2007

  • McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
  • Journal of Applied Crystallography, Vol. 40, Issue 4
  • DOI: 10.1107/S0021889807021206

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Refinement of Macromolecular Structures by the Maximum-Likelihood Method
journal, May 1997

  • Murshudov, G. N.; Vagin, A. A.; Dodson, E. J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 53, Issue 3
  • DOI: 10.1107/S0907444996012255

Overview of the CCP 4 suite and current developments
journal, March 2011

  • Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444910045749

VMD: Visual molecular dynamics
journal, February 1996


Web 3DNA--a web server for the analysis, reconstruction, and visualization of three-dimensional nucleic-acid structures
journal, May 2009

  • Zheng, G.; Lu, X. -J.; Olson, W. K.
  • Nucleic Acids Research, Vol. 37, Issue Web Server
  • DOI: 10.1093/nar/gkp358

Digital detection of endonuclease mediated gene disruption in the HIV provirus
journal, February 2016

  • Sedlak, Ruth Hall; Liang, Shu; Niyonzima, Nixon
  • Scientific Reports, Vol. 6, Issue 1
  • DOI: 10.1038/srep20064

Tapping natural reservoirs of homing endonucleases for targeted gene modification
journal, July 2011

  • Takeuchi, Ryo; Lambert, Abigail R.; Mak, Amanda Nga-Sze
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 32
  • DOI: 10.1073/pnas.1107719108

On the possibilities and limitations of rational protein design to expand the specificity of restriction enzymes: a case study employing EcoRV as the target
journal, April 2000

  • Lanio, Thomas; Jeltsch, Albert; Pingoud, Alfred
  • Protein Engineering, Design and Selection, Vol. 13, Issue 4
  • DOI: 10.1093/protein/13.4.275

Engineering novel restriction endonucleases: Principles and applications
journal, July 1996


Type II restriction endonucleases—a historical perspective and more
journal, May 2014

  • Pingoud, Alfred; Wilson, Geoffrey G.; Wende, Wolfgang
  • Nucleic Acids Research, Vol. 42, Issue 12
  • DOI: 10.1093/nar/gku447

Recognizing DNA
journal, November 2005


Engineered Protein Machines: Emergent Tools for Synthetic Biology
journal, January 2016


    Works referencing / citing this record:

    ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges
    journal, January 2020


    Gene drives to fight malaria: current state and future directions
    journal, November 2017


    Engineering altered protein–DNA recognition specificity
    journal, April 2018

    • Bogdanove, Adam J.; Bohm, Andrew; Miller, Jeffrey C.
    • Nucleic Acids Research, Vol. 46, Issue 10
    • DOI: 10.1093/nar/gky289