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.
Highlights
Homing endonucleases (HEs) are sequence-specific enzymes that recognize and cleave DNA at long target sites
Using a yeast surface display high-throughput cleavage selection system [9], here we show the application of rational design with directed evolution in a progressive approach to achieve a large scale re-engineering of the I-Anil homing endonuclease to recognize a unique sequence in the mouse Bruton tyrosine kinase (Btk) gene differing by 9 bp from the native I-AniI sequence
Human X-linked agammaglobulinemia (XLA) is a rare X-linked genetic disorder caused by mutations in the human BTK gene, which is expressed at all stages of B-lineage development and is required for pre-B cell expansion and mature B cell survival and activation [17,18]
Summary
Homing endonucleases (HEs) are sequence-specific enzymes that recognize and cleave DNA at long target sites (typically 20 bp) They are typically encoded within introns or inteins, and behave as mobile genetic elements that copy their genetic information into intron- or intein-less alleles of their host gene. A variety of approaches have been applied to generate LHE variants with new cleavage specificities, most of them involving ‘local’ variant library generation through random mutation or structural-based modification of the LHE protein interface that contacts the DNA target site, followed by selection based on DNA cleavage or recombination activities [5,6,7,8] These methods currently are able to generate variants with changes in cleavage specificity in a ‘local’ region of the LHE DNA/protein interface covering a relatively small number of contiguous base pairs. The development of methods to overcome limitations in large scale LHE re-specification is necessary to ex-
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