Natural and Engineered Gene Targeting Proteins

Genome engineering and targeted gene modification is a rapidly maturing discipline in which genomes within cell lines, tissues or organisms are manipulated and altered at specified individual loci. Such approaches are now being used for a wide-variety of purposes, including:

  • Gene correction in patients suffering from genetic diseases.
  • Targeted disruption of genes in patients afflicted by latent viral infections.
  • The modification or insertion of genes in plants.
  • The generation of transgenic stem cell lines.
  • The genetic modification of animal models.
  • The incorporation of gene drive systems into disease vectors as part of population control strategies.

A variety of systems are being developed for this purpose, including site-specific DNA cleaving enzymes. These systems induce double strand breaks that are repaired via homologous recombination and/or nonhomologous end joining, resulting in targeted gene modification and disruption, respectively.

Our laboratory has worked to determine the structure, function and mechanism of homing endonucleases, rare-cutting endonucleases and gene targeting proteins since 1996.   Since that time, we have solved the first (and in many cases, still the only) structures of representative members of most of the known families of homing endonucleases.  Subsequently, we reported the first examples of structure-based redesign of homing endonucleases that recognize and cut non-native target sites.  More recently, we reported comprehensive comparison of binding and cleavage specificity profiles of individual homing endonucleases, as part of ongoing directed evolution and engineering experiments.  Our studies of LHEs have provided the basis for extensive research and development by several biotech companies that engineer LAGLIDADG homing endonucleases for gene targeting.  

Homing Endonuclease Structures

HEs have evolved independently multiple times, in microbes and phage common to every known biological kingdom.  Each of the structures shown (image, right) correspond to representative HEs from these kingdoms, as determined in the Stoddard laboratory

Above left: I-HmuI is an HNH homing endonuclease, which are found in bacteriophage (along with GIY-YIG HEs).  Structure solved by Betty Shen, in collaboration with the David Shub Laboratory at SUNY-Albany.

Above Right:  I-Ssp6803I is a PD...(D/E)xK homing endonuclease, which are found in cyanobacteria.  Structure solved by Lei Zhao, again in collaboration with the David Shub Laboratory at SUNY-Albany.

Lower left: I-AniI is a LAGLIDADG homing endonuclease, which are found in the organellar genomes of eukarya and in archaea.  Structure solved by Jill Bolduc, in collaboration with Mark Caprara (Case Western University) and Richard Waring (Temple University)

Lower right:  I-PpoI is a His-Cys box homing endonuclease, which are found in protists.  Structure solved by Karen Flick, in collaboration with the Ray Monnat Laboratory (University of Washington).

Homing Endonuclease Structures
Crystallographic structure of the DNA binding region of the PthXo1 TAL effector.

More recently, our laboratory has solved a structure of a  Transcription Activator Like ('TAL') effector bound to its DNA target site.  These proteins, from which gene targeting endonucleases (TAL endonucleases or 'TALENs') can be derived, are encoded by pathogenic bacteria (Xanthomonas and Ralstonia) that infect a wide range of plants, including most important agricultural cultivars.  The repetitious modular architecture of TALs facilitates their rapid and efficient engineering and application for any of the types of genome engineering described above.

(Image, left) Crystallographic structure of the DNA binding region of the PthXo1 TAL effector. This naturally occurring TAL effector is encoded by Xanthomonas oryzae, and binds a single genomic target site in the rice genome. Structure solved by Amanda Mak in collaboration with Phil Bradley (FHCRC Computational Biology Program) and Adam Bogdanove (Cornell University, Ithaca).


Our current work in this field continues to be evenly divided between fundamental studies of gene targeting protein structures and function, and the development and application of broadly generalizable methods for the retargeting of gene targeting protein scaffolds, both for genome engineering (in the form of new methods for targeted gene insertions or gene knockout/knock-in experiments) and for corrective gene therapy. 

A major focus is on the creation of a new generation of 'hyper-specific' gene targeting nucleases (termed 'MegaTALs') that can be safely used for human gene therapy in combination with stem cell replacement approaches.

We also continue to develop LAGLIDADG homing endonucleases ('LHEs', or 'Meganucleases') as a resource for the creation of site-specific endonuclease scaffolds.  Much of that work is databased in the LAHEDES webserver.