Beschreibung:
In the developing field of genomic engineering, several strategies for targeted, specific genetic modification have recently been developed. Many current methods, such as CRISPR, utilize double strand breaks (DSBs) in DNA to initiate an innate repair process called homology directed repair (HDR). DSBs can also trigger another repair pathway, non‐homologous end joining (NHEJ). However, NHEJ has a lower fidelity and can lead to harmful mutations or translocations. Single strand breaks (SSBs) in DNA stimulate HDR while avoiding the NHEJ pathway. Nickases are enzymes that are capable of inducing SSBs in DNA via a single cut, or nick, in one strand of DNA. Therefore, nickases are ideal for developing targeted and specific genetic modifications.LAGLIDLAG homing endonuclease (LHEs) are monomeric enzymes with long recognition sequences; these properties make then good candidates for conversion to nickases. In collaboration with Dr. Lori Robins (University of Washington Bothell), we are carrying out a structure‐guided mutagenesis of various LHEs with the goal of converting them into functional nickases. Two LHEs, LtrWI and SmaMI, have been chosen to mutate into functional nickases. Site‐directed mutagenesis of these LHE genes is aimed at altering single amino acids that are believed to catalyze DNA hydrolysis in one of the active sites. By inactivating one of the active sites in the monomer and allowing the other active site to remain intact, this produces a semi‐functional enzyme capable of nicking DNA at specific sequences.Through PCR‐directed mutagenesis of specific active‐site lysine residues, we have successfully produced LtrWI and SmaMI mutants with predominately nicking activity. Current efforts seek to extend these observations to additional LHEs, through identification and mutagenesis of homologous lysine residues in the cognate active sites.Support or Funding InformationThank you to the Cal Poly Student based fee and Chevron Applied Biotechnology Research Program for funding; Ellen Calcagno for administrative assistance and Michael Black for technical assistance; members of the Hillers/Turner lab for helpful discussion and support; Conor Beck for diagrammatic interpretation of data.