Advertisement

 

 

Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method.

Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method.
Author Information (click to view)

Zotova A, Lopatukhina E, Filatov A, Khaitov M, Mazurov D,


Zotova A, Lopatukhina E, Filatov A, Khaitov M, Mazurov D, (click to view)

Zotova A, Lopatukhina E, Filatov A, Khaitov M, Mazurov D,

Advertisement

Viruses 2017 11 029(11) pii E325
Abstract

Programmable endonucleases introduce DNA breaks at specific sites, which are repaired by non-homologous end joining (NHEJ) or homology recombination (HDR). Genome editing in human lymphoid cells is challenging as these difficult-to-transfect cells may also inefficiently repair DNA by HDR. Here, we estimated efficiencies and dynamics of knockout (KO) and knockin (KI) generation in human T and B cell lines depending on repair template, target loci and types of genomic endonucleases. Using zinc finger nuclease (ZFN), we have engineered Jurkat and CEM cells with the 8.2 kb human immunodeficiency virus type 1 (HIV-1) ∆Env genome integrated at the adeno-associated virus integration site 1 (AAVS1) locus that stably produce virus particles and mediate infection upon transfection with helper vectors. Knockouts generated by ZFN or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) double nicking techniques were comparably efficient in lymphoid cells. However, unlike polyclonal sorted cells, gene-edited cells selected by cloning exerted tremendous deviations in functionality as estimated by replication of HIV-1 and human T cell leukemia virus type 1 (HTLV-1) in these cells. Notably, the recently reported high-fidelity eCas9 1.1 when combined to the nickase mutation displayed gene-dependent decrease in on-target activity. Thus, the balance between off-target effects and on-target efficiency of nucleases, as well as choice of the optimal method of edited cell selection should be taken into account for proper gene function validation in lymphoid cells.

Submit a Comment

Your email address will not be published. Required fields are marked *

4 × two =

[ HIDE/SHOW ]