Engineering Meganuclease for Precise Plant Genome Modification

Chapter 2 Engineering Meganuclease for Precise Plant Genome Modifi cation Fayza Daboussi , Thomas J. Stoddard , and Feng Zhang Abstract Meganucleases, also termed homing endonucleases, are rare-cutting enzymes that are encoded within the genome of nearly all microbes. These enzymes recognize and cleave long DNA sequences (typically 18–30 base pairs) generating double-strand DNA breaks (DSBs). The resulting DSBs can be repaired by different pathways leading to a variety of site-specifi c DNA modifi cations, such as insertions, deletions, or point mutations. Over the past 15 years tremendous efforts have been made to engineer a number of variant meganucleases that cleave novel DNA targets. Engineered meganucleases are now being used to generate targeted genomic modifi cations for a variety of basic and biotechnology applications, including creating valuable traits in crop species. 1 Introduction The burgeoning demand for plant-derived products, such as food, feed, fuel, and fi ber, underlies the importance of developing methods to continuously improve crop varieties with higher yields, lower input costs, and better nutritional value. A variety of technologies have been developed to enable creation of genetic variations with desirable traits. Approaches using mutagens such as ethyl methanesulfonate, gamma rays, fast neutron, and transfer DNA (T-DNA) involves the random introduction of mutations into the genome followed by large-scale screens of mutagenized popula- tions. These methods largely depend on chance and have nearly no control over location and types of mutations recovered from the population. It has been a long sought-after goal to develop methods that could generate specifi c and targeted genetic mutations in plants. With more than 50 plant genomes being sequenced, genome modifi cation approaches with improved precision will greatly facilitate the dissection of gene function, as well as creating new traits in both basic and applied research (Michael and Jackson 2013 ; Voytas 2013 ). One of the most powerful means to introduce specifi c changes into genomes is through double-strand DNA break (DSB) repair pathways. Two major pathways, F. Daboussi • T. J. Stoddard , B.Sc. • F. Zhang , Ph.D. (*) Cellectis Plant Sciences Inc. , 600 County Road D, Suite 8 , New Brighton , MN 55112 , USA e-mail: [email protected]; [email protected] © Springer Science+Business Media New York 2015 21 F. Zhang et al. (eds.), Advances in New Technology for Targeted Modifi cation of Plant Genomes, DOI 10.1007/978-1-4939-2556-8_2 22 F. Daboussi et al. homologous recombination (HR) and nonhomologous end joining (NHEJ), have been employed to incorporate mutations at the break site (Voytas 2013 ; Puchta and Fauser 2013 ). In order to utilize DNA repair pathways, targeted DSBs fi rst have to be created in the desired location. Efforts have been focused on developing sequence- specifi c nucleases that could be engineered to create DSBs in a target locus. To date, four classes of sequence-specifi c nucleases, meganucleases (homing endonuclease), zinc fi nger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), can be chosen to cleave almost any sequence in any species. While ZFNs, TALENs, and CRISPR/Cas9 technologies are reviewed in other chapters, this review focuses on the state of the art in meganuclease engineering and its applications in plant genome engineering (Voytas 2013 ; Puchta and Fauser 2013 ; Small and Puchta 2014 ). Meganucleases were the fi rst class of sequence-specifi c nucleases employed to create targeted DSBs in eukaryote genomes. The very fi rst meganuclease, I - SecI , was discovered and characterized from yeast in the 1970s and 1980s (Faye et al. 1979 ; Bos et al. 1978 ; Jacquier and Dujon 1985 ). Encoded by an intron in the mito- chondrial large ribosomal RNA subunit (LsrRNA), this enzyme recognizes and cleaves an 18 bp unsymmetrical sequence in an intron-free allele of the LsrRNA gene (Jacquier and Dujon 1985 ; Stoddard 2014 ). The resulting break is repaired via HR using the intron-harboring allele as a repair template leading to insertion of the intron into the target site, this process is known as intron homing. Because of its long DNA recognition sequence and high specifi city, I - SceI can be readily expressed in higher organisms without cleaving host genomes and inducing cytotoxicity. For these reasons it has been used to introduce DSBs at defi ned locations in studies of DNA repair mechanisms in many eukaryote genomes (Choulika et al. 1995 ; Rouet et al. 1994a , b ). In 1993, Puchta et al. fi rst demonstrated that DSBs induced by I - SceI enhance HR in plant species, namely Nicotiana tabacum (Puchta 1999 ; Puchta et al. 1993 ). This landmark research heralded the arrival of precise genome engineering in plants using sequence-specifi c nucleases. Use of meganucleases in precise genome modifi cation requires the ability to engineer nucleases with new sequence specifi city. One challenge of meganuclease engineering is that cleavage and DNA-binding domains overlap (Stoddard 2011 ). When an amino acid sequence is altered to achieve new DNA sequence specifi city, the catalytic activity of the enzyme is often compromised. Despite these diffi culties, advances in high-throughput screening and protein modeling have made meganucleases easier to engineer and more accessible. This review will discuss the latest developments in customized meganucleases, the strategies to increase meganuclease activity, and the recent applications of engineered meganucleases in plant genome modifi cation. 2 Engineering of Meganucleases for New Sequence Specifi city Although the discovery of the meganuclease dates back to the 1970s, widespread use as tools for genome editing has been hampered by the lack of methods allowing for effi cient protein engineering of the protein–DNA interface (Stoddard 2014 ). 2 Engineering Meganuclease for Precise Plant Genome Modifi cation 23 In the 2000s, a semi-rational approach based on computational analysis of structural features in the protein–DNA interface has emerged as a promising strategy to generate meganucleases with new specifi cities (Chevalier et al. 2002 ; Ashworth et al. 2006 ). This approach allowed for mutating specifi c residues that led to a signifi cant decrease in the complexity of the mutant library to be processed. Using I - CreI as a model scaffold, several groups have been able to alter its DNA recognition properties on both a small and large scale, in terms of total proteins reengineered. Initial successes with the I - CreI (Seligman et al. 2002 ; Sussman et al. 2004 ; Rosen et al. 2006 ) and I - SceI scaffolds (Gimble et al. 2003 ; Chen and Zhao 2005 ) that used small-scale libraries resulted in the identifi cation of only a few mutants. Later, Seligman and co-workers demonstrated that substitution of specifi c individual residues in the I - CreI αββαββα fold induces substantial cleavage of novel targets (Seligman et al. 2002 ). Then Gimble and collaborators modifi ed the DNA-binding domain of PI - SceI and selected variant proteins with altered binding specifi city using a double-hybrid binding assay (Gimble et al. 2003 ). While being able to generate a few meganucleases with novel DNA binding specifi city, all attempts were limited to modifi cation of a small subset of amino acid. Recently several large-scale screening approaches, including the one developed by our group, has been reported to modulate the DNA-binding and catalytic properties of meganucleases (Jarjour et al. 2009 ; Volna et al. 2007 ; Baxter et al. 2013 ). The method implemented in our group, termed the two-step semi-rational approach, enables the complete redesign of endonuclease specifi city. In this method, the fi rst step consists of randomizing specifi c residues in the DNA-binding domain of the protein and identifying collections of variants with locally altered specifi city. The second step, namely the combinatorial step, is to combine and assemble sets of mutants from different locally engineered variants to create globally engineered proteins with predictable specifi city. Using this method, thousands of I - CreI mutants with locally altered specifi cities have been identifi ed in our databank (Epinat et al. 2003 ; Arnould et al. 2006 ; Smith et al. 2006 ). 2.1 Yeast High-Throughput Screening Assay In order to screen a large number of nuclease variants in the fi rst step, an automated high-throughput screening method has been developed in Saccharomyces cerevisiae (Epinat et al. 2003 ; Chames et al. 2005 ). As shown in Fig. 2.1 , two strains of S. cerevisiae were used for the screening of meganuclease activity: the fi rst strain, MAT α drives expression of the meganuclease with a galactose-inducible promoter; while the second strain, MAT a, carries the substrate composed of an inactive LacZ gene under the control of a constitutive promoter. The LacZ gene is interrupted by an intervening sequence that is fl anked by two direct repeats containing the target of interest and an I - SceI target site as an internal control. Upon mating of the two strains, diploid cells were selected with the help of the auxotrophic markers present in both meganuclease and LacZ expression vectors. These strains were then trans- ferred onto galactose-rich medium, inducing meganuclease expression. The expressed meganucleases will introduce a DSB at their target leading to the 24 F. Daboussi et al. Fig. 2.1 Yeast high-throughput screening assay. One yeast strain, MAT α, contains the meganuclease whose expression is driven by a galactose-inducible promoter. Another strain, MATa , contains a single-strand annealing (SSA) reporter vector composed of the LacZ gene interrupted by the target of interest and an I - SceI target site as an internal control, fl anked by two direct repeat. After mating and transformed diploid selection, the expression of the enzyme is induced on galactose- rich medium.

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