title: Progressive engineering of a homing endonuclease genome editing reagent for the murine X-linked immunodeficiency locus.
abstract: LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20-22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)-a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering.
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Yupeng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Iram F
authors.initials: IF
authors.lastname: Khan
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Boissel
authors.affiliation: Pregenen Inc., Seattle, WA 98103, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Jarjour
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Pangallo
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Summer
authors.initials: S
authors.lastname: Thyme
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA Departments of Pediatrics and Immunology, University of Washington, Seattle, WA 98195, USA drawling@u.washington.edu.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
authors.affiliation: Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA Departments of Pediatrics and Immunology, University of Washington, Seattle, WA 98195, USA drawling@u.washington.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rawlings
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D002478:Cells, Cultured; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry; D019020:Directed Molecular Evolution; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D056426:Genetic Loci; D023281:Genomics; D057809:HEK293 Cells; D059765:Homologous Recombination; D006801:Humans; D007202:Indicators and Reagents; D051379:Mice; D009154:Mutation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000625951:Btk protein, mouse; D004706:Endodeoxyribonucleases