Prediction of Talen Binding Site for Genome Editing in Stem Cells

Abstract

Transcription activator-like effector (TALE) nucleases (TALENs) have currently risen as a genome editing tool in numerous live forms .Transcription activator-like effector nucleases (TALENs), comprising of an DNA binding domain and a FokI cleavage domain TALENs can behaves as adaptable molecular DNA scissors creating double-strand breaks (DSBs) at desired position of a genome, expanding the effectiveness of genomic modification. The TALE central repeat domains’s flexible nature empower to tailor DNA recognition specificity easily and target basically any desired DNA sequence. In this study we understood the structural basis of interaction between TALEN and DNA and the target binding site of TALEN was predicted. It was found that NH RVD binds Guanine more specifically than NN RVD and accordingly script of TALE NT TOOL 2.0(https://tale-nt.cac.cornell.edu/node/add/talen) was modified. Adh2 and adh1c gene were tested in both the existing and modified script of TALE NT tool 2.0. Experiment shows that a 63 aa CTS in TALEN scaffold ,could lead to effective gene modification in cells of human, when separated by 14-32 bp spacers and accordingly script of TALE NT was changed with the spacer length 15-30.The results of adh2 gene and adh1c gene were verified by online available tools of TALEN LIBRARY RESEARCH (http://www.talenlibrary.net/) and we got affirmative results. Thus, the modified script is expected to increase the efficiency of DNA targeting.