Re-engineering genomes using single stranded oligonucleotides and TAL-

effector Nucleases (TALENs) for industrial technology

Dr. Eric B. Kmiec

Center for Translational Cancer Research Helen F. Graham Cancer Center

Christiana Hospitaland

Delaware State University

Pure basic research(Bohr)

Use-inspired basic research(Pasteur)

Pure applied research(Edison)

“Pasteur’s Quadrant”

High

High

Low

Low

Quest for Fundamental

Understanding

Consideration of UseStokes, Donald. Pasteur's Quadrant Basic Science and Technology Innovation. Washington, DC: Brookings Institution Press, 1997. Print.

ssODN

ZFN

TALEN

Technologies and Tools

Gene Editing

CRISPR/Cas9

Kim, H., Kim, J.  A guide to genome engineering with programmable nucleases.  Nature Review Genetics doi:10.1038/nrg3686 (2014)

Kim, H., Kim, J.  A guide to genome engineering with programmable nucleases.  Nature Review Genetics doi:10.1038/nrg3686 (2014)

Kim, H., Kim, J.  A guide to genome engineering with programmable nucleases.  Nature Review Genetics doi:10.1038/nrg3686 (2014)

Kim, H., Kim, J.  A guide to genome engineering with programmable nucleases.  Nature Review Genetics doi:10.1038/nrg3686 (2014)

Kim, H., Kim, J.  A guide to genome engineering with programmable nucleases.  Nature Review Genetics doi:10.1038/nrg3686 (2014)

Kim, H., Kim, J.  A guide to genome engineering with programmable nucleases.  Nature Review Genetics doi:10.1038/nrg3686 (2014)

Gene-Editing Model System

Regulation of Gene Editing

Oligonucleotide Enters Cell

Aligns in Homologous Register at Target Site

Creates D-loop

DNA Replication Activity

Transcriptional Activity

Homeologous Recombination

Activates DNA Damage Response Pathway

p53P

ATMP/ATRP

CHK1/CHK2P

Activation

Cell Cycle Delay (S)S phase stalling

H2AXP DS-BreakRepair

Coordinates Repair and Restart of Fork

Replication stallingFork Collapse

DS-Breaks

Incorporates into Target Site : replaces mutant

sequence

Gene conversion via Transcription-Coupled Repair

Gene conversion via Mismatch Repair

DNA Damage Elevates Gene

Editing Frequency

BLMHelicase

and/or

and/or

DNA Pairing Activity

RAD51(etc)

CellSurvival

The Kmiec Laboratory

2000-2011 Graduate StudentsHetal Parekh-Olmedo, Ph.D.Erin Brachman, Ph.D.Miya Drury, Ph.D.Luciana Ferrara, Ph.D.Katie McGuire, Ph.D.Julia Engstrom, Ph.D.Jennifer Sonntag, Ph.D.Kerry Falgowski, Ph.D.Jennifer Illuzzi, Ph.D.Melissa Bonner, Ph.D.Timothy Swartz, Ph.D.

Rohina NiamatBryan StrousePawel BialkNatalia RiveraShani SamuelBrett Sansbury

1995-2000 Graduate StudentsJoanne Sekiguchi, Ph.D.Allyson Cole-Strauss, Ph.D.Hideohito Kotani, Ph.D. Seema Dutta, Ph.D. Ronald Myers, Ph.D.

Postdoctoral FellowsDavid Gerhold,Ph.D.Kyggeon Yoon, Ph.D.

Postdoctoral FellowsSubhais Sandu, Ph.D.Dula Man, Ph.D