Evaluating different DNA binding domains to modulate L1 ORF2p-driven site-specific retrotransposition events in human cells.

Evaluating different DNA binding domains to modulate L1 ORF2p-driven site-specific retrotransposition events in human cells.
Gene. 2017 Nov 14;:
Authors: Ade CM, Derbes RS, Wagstaff BJ, Linker SB, White TB, Deharo D, Belancio VP, Ivics Z, Roy-Engel AM
Abstract
DNA binding domains (DBDs) have been used with great success to impart targeting capabilities to a variety of proteins creating highly useful genomic tools. We evaluated the ability of five types of DBDs and strategies (AAV Rep proteins, Cre, TAL effectors, zinc finger proteins, and Cas9/gRNA system) to target the L1 ORF2 protein to drive retrotransposition of Alu inserts to specific sequences in the human genome. First, we find that the L1 ORF2 protein tolerates the addition of protein domains both at the amino- and carboxy-terminus. Although in some instances retrotransposition efficiencies slightly diminished, all fusion proteins containing an intact ORF2 were capable of driving retrotransposition. Second, the stability of individual ORF2 fusion proteins varies and difficult to predict. Third, DBDs that require the formation of multimers for target recognition are unlikely to modify targeting of ORF2-driven insertions. Fourth, the more components needed to assemble into a complex to drive targeted retrotransposition, the less likely the strategy will increase targeted insertions. Fifth, abundance of target sequences present in the genome will likely dictate the effectiveness...

Source: Gene - November 14, 2017 Category: Genetics & Stem Cells Authors: Ade CM, Derbes RS, Wagstaff BJ, Linker SB, White TB, Deharo D, Belancio VP, Ivics Z, Roy-Engel AM Tags: Gene Source Type: research