Gene editing in human lymphoid cells: Role for donor DNA, type of genomic nuclease and cell selection method

Anastasia Zotova, Elena Lopatukhina, Alexander Filatov, Musa Khaitov, Dmitriy Mazurov

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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) DEnv 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.

Original languageEnglish (US)
Article number325
JournalViruses
Volume9
Issue number11
DOIs
StatePublished - Nov 2017
Externally publishedYes

Bibliographical note

Funding Information:
Acknowledgments: This work has been supported by the grants of the Russian Science Foundation (RSF 15-15-00135), and partially by the Russian Foundation for Basic Research (RFBR 15-04-04364). We are grateful to Alexey Pichugin from the Insitute of Immunology for the assistance in cell sorting, Mikhail Pashenkov from the Institute of Immunology, and William Telford from NIH (Bethesda) for the critical reading of the manuscript.

Publisher Copyright:
© 2017 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

  • Cell functionality
  • Cell sorting
  • CRISPR-Cas9
  • Gene editing
  • HIV-1
  • Knockin
  • Knockout
  • T lymphocytes
  • ZFN

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