Compact designer TALENs for efficient genome engineering

Transcription activator-like effector nucleases are readily targetable ‘molecular scissors’ for genome engineering applications. These artificial nucleases offer high specificity coupled with simplicity in design that results from the ability to serially chain transcription activator-like effector repeat arrays to target individual DNA bases. However, these benefits come at the cost of an appreciably large multimeric protein complex, in which DNA cleavage is governed by the nonspecific FokI nuclease domain. Here we report a significant improvement to the standard transcription activator-like effector nuclease architecture by leveraging the partially specific I-TevI catalytic domain to create a new class of monomeric, DNA-cleaving enzymes. In vivo yeast, plant and mammalian cell assays demonstrate that the half-size, single-polypeptide compact transcription activator-like effector nucleases exhibit overall activity and specificity comparable to currently available designer nucleases. In addition, we harness the catalytic mechanism of I-TevI to generate novel compact transcription activator-like effector nuclease-based nicking enzymes that display a greater than 25-fold increase in relative targeted gene correction efficacy.

[1]  S Chandrasegaran,et al.  Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Erin L. Doyle,et al.  Targeting DNA Double-Strand Breaks with TAL Effector Nucleases , 2010, Genetics.

[3]  G. Church,et al.  Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. , 2011, Nature biotechnology.

[4]  M. Isalan.  Zinc-finger nucleases: how to play two good hands , 2011, Nature Methods.

[5]  J. Sheen,et al.  Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis , 2007, Nature Protocols.

[6]  P. Duchateau,et al.  Meganucleases and Other Tools for Targeted Genome Engineering: Perspectives and Challenges for Gene Therapy , 2011, Current gene therapy.

[7]  Sarah K. Baxter,et al.  Coupling endonucleases with DNA end–processing enzymes to drive gene disruption , 2012, Nature Methods.

[8]  Jens Boch,et al.  Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors , 2009, Science.

[9]  Huimin Zhao,et al.  Review Stepping Stones in Dna Sequencing Review Recent Advances in Targeted Genome Engineering in Mammalian Systems Review Glyco-engineering in Plants to Produce Human-like N-glycan Structures Review Using Storage Organelles for the Accumulation and Encapsulation of Recombinant Proteins the Challeng , 2022 .

[10]  Matthew J. Moscou,et al.  A Simple Cipher Governs DNA Recognition by TAL Effectors , 2009, Science.

[11]  P. Duchateau,et al.  A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences , 2006, Nucleic acids research.

[12]  N. Maizels,et al.  DNA Nicks Promote Efficient and Safe Targeted Gene Correction , 2011, PloS one.

[13]  A. Pingoud,et al.  Generation of Novel Nucleases with Extended Specificity by Rational and Combinatorial Strategies , 2011, Chembiochem : a European journal of chemical biology.

[14]  T. Lahaye,et al.  Assembly of custom TALE-type DNA binding domains by modular cloning , 2011, Nucleic acids research.

[15]  Jens Boch,et al.  TAL effector RVD specificities and efficiencies , 2012, Nature Biotechnology.

[16]  Fayza Daboussi,et al.  Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation*♦ , 2012, The Journal of Biological Chemistry.

[17]  David Baker,et al.  Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases , 2012, Nucleic acids research.

[18]  Mark Isalan,et al.  Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  David R. Liu,et al.  Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection , 2011, Nature Methods.

[20]  Susan Carpenter,et al.  Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes , 2011, Nucleic acids research.

[21]  Nieng Yan,et al.  Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors , 2012, Science.

[22]  Hicham Mansour,et al.  Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein , 2011, Plant Molecular Biology.

[23]  Jeffrey C. Miller,et al.  An unbiased genome-wide analysis of zinc-finger nuclease specificity , 2011, Nature Biotechnology.

[24]  M. Belfort,et al.  Zinc finger as distance determinant in the flexible linker of intron endonuclease I-TevI , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Claudio Mussolino,et al.  A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity , 2011, Nucleic acids research.

[26]  Claudio Mussolino,et al.  Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects , 2012, Nucleic acids research.

[27]  T. Cathomen,et al.  Zinc-finger nuclease based genome surgery: it's all about specificity. , 2011, Current gene therapy.

[28]  Elo Leung,et al.  A TALE nuclease architecture for efficient genome editing , 2011, Nature Biotechnology.

[29]  Jeffrey J. Gray,et al.  Targeted DNA Methylation Using an Artificially Bisected M.HhaI Fused to Zinc Fingers , 2012, PloS one.

[30]  P. Gregory,et al.  Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors , 2007, Gene Therapy.

[31]  B. Kleinstiver,et al.  Monomeric site-specific nucleases for genome editing , 2012, Proceedings of the National Academy of Sciences.

[32]  Alfred Pingoud,et al.  Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases , 2011, Nucleic acids research.

[33]  Jens Boch,et al.  Transcriptional Activators of Human Genes with Programmable DNA-Specificity , 2011, PloS one.

[34]  Alfred Pingoud,et al.  A novel zinc-finger nuclease platform with a sequence-specific cleavage module , 2011, Nucleic acids research.

[35]  George H. Silva,et al.  High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes , 2013, PloS one.

[36]  C. Proudfoot,et al.  Zinc Finger Recombinases with Adaptable DNA Sequence Specificity , 2011, PloS one.

[37]  Marc Ostermeier,et al.  Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells , 2009, Nucleic acids research.

[38]  Daniel F. Voytas,et al.  Targeting G with TAL Effectors: A Comparison of Activities of TALENs Constructed with NN and NK Repeat Variable Di-Residues , 2012, PloS one.

[39]  P. Duchateau,et al.  Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. , 2006, Journal of molecular biology.

[40]  J. E. Mueller,et al.  Intron‐encoded endonuclease I‐TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site. , 1995, The EMBO journal.

[41]  F. J. Olorunniji,et al.  Zinc-finger recombinase activities in vitro , 2011, Nucleic acids research.

[42]  I. Ial,et al.  Nature Communications , 2010, Nature Cell Biology.

[43]  Erin L. Doyle,et al.  Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting , 2011, Nucleic acids research.

[44]  M. Belfort,et al.  Coincidence of cleavage sites of intron endonuclease I-TevI and critical sequences of the host thymidylate synthase gene. , 2004, Journal of molecular biology.

[45]  Jens Boch,et al.  Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors , 2010, Proceedings of the National Academy of Sciences.

[46]  B. Stoddard,et al.  Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template , 2010, Nucleic acids research.

[47]  Philip Bradley,et al.  The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target , 2012, Science.

[48]  Jeffry D Sander,et al.  FLAsH assembly of TALeNs for high-throughput genome editing , 2022 .

[49]  Ruhong Zhou,et al.  Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains , 2012, Nature Communications.