Efficient genome engineering in mammalian systems using TAL effector nucleases and CRISPR/Cas system

Abstract

Programmable nucleases like transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) are broadly used for engineering of genome in various cells and organisms. TALENs are recently developed fusion proteins of a modular DNA-binding TALE repeat domain and a catalytic nuclease domain of FokI restriction endonuclease. In this thesis, I designed and synthesized highly active TALENs to target the progesterone immunomodulatory binding factor 1 (Pibf1) and the selenoprotein W, muscle 1 (Sepw1) gene. Non-homologous end-joining (NHEJ) mediated indel mutations were detected in transfected mouse cells with plasmids encoding these TALENs and knock-out mice of these genes were successfully generated via embryo microinjection. RGENs are efficient artificial endonucleases using CRISPR/Cas system which is a prokaryotic immune system. In this study, I have designed RGENs to target inverted region of the blood coagulation factor VIII (F8) gene that is known as a major cause of severe hemophilia phenotype. When two RGENs were used to generate double-strand breaks (DSB) at their endogenous target loci in wild-type Hela cells, the large chromosomal segment up to 600-kbp is inverted. Furthermore, the inverted region of induced pluripotent stem cells (iPSC) from hemophilia patients is also successfully reverted via these RGENs and mRNA expression of F8 is recovered. TALENs and RGENs have enabled targeted genome editing easily and efficiently. These technologies will be very useful systems for research of gene functions and correcting the genetic causes of many diseases.