ISSN 1866-8836
Клеточная терапия и трансплантация

GC-02. Optimization of genome editing conditions in hematopoietic stem cells using designer nucleases TALEN and CRISPR/Cas9

Kirill V. Lepik1, Alyona I. Shakirova1, Marina О. Popova1, Vladislav S. Sergeev1, Albert R. Muslimov1, Boris Fehse2, Alexander D. Kulagin1

1 RM Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantation, Pavlov University, St. Petersburg, Russia
2 Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Contact: Dr. Kirill V. Lepik, e-mail:

doi 10.18620/ctt-1866-8836-2020-9-3-1-152



Gene therapy is a promising treatment approach for a number of hereditary and acquired diseases that are currently considered incurable. Genome editing technology opens up perspectives for introduction of stable genetic modifications in the desired loci. Hematopoietic stem cells (HSCs) are a classic target for therapeutic genome modifications ex vivo, with the potential to achieve a persistent phenotype correction in severe combined immunodeficiencies, hemoglobinopathies, storage diseases (i.e. Hurler’s syndrome, adrenoleukodystrophy) and socially significant diseases, including HIV infection. The aim of this study is to refine the protocol and optimize the conditions for genome editing in primary human HSCs using TALEN and CRISPR/Cas9 designer nucleases.

Materials and methods

The study was carried out in accordance with the Helsinki declaration. All donors of biological material gave informed voluntary consent to participate in the study. HSCs were isolated from the bone marrow of healthy donors by immunomagnetic separation using the CD34 MicroBead kit (Miltenyi Biotec). In vitro transcription of mRNA for eGFP protein, as well as TALEN and Cas9, was performed using the T7-mScript Standard system (Biozym). In vitro transcription CCR5 specific RNAs (gRNAs) was performed using the MEGAshortscript T7 Transcription Kit reagent kit (Thermofisher). After HSC preactivation, transfection of eGFP, TALEN mRNA cells or a mixture of Cas9 mRNA with CCR5-specific gRNAs was performed using the Gene Pulser Xcell electroporator (BioRad) with different electroporation parameters: the number (1 or 2) and pulse characteristics (voltage range 220-500V, pulse duration 5-10 ms). Transfection efficiency was analyzed by assessment of the number of GFP positive and viable cells using flow cytometry. Editing efficiency was evaluated using digital droplet PCR (Bio-Rad) or Surveyor nuclease (SN) assay (EnGen® Mutation Detection Kit, NEB).


Enrichment of the CD34 + HSC population after selection was at least 85% (85-98%). The analysis of HSC transfection efficiency at various electroporation parameters (220V/38ms, 220V/10ms, 2×220V/10ms, 300V/5ms, 2×300V/5ms, 300V/10ms, 500V/5ms, 500V/10ms), demonstrated different transfection rates with 35%-80% of GFP+CD34+ cells, as well as a decrease in the number of viable HSCs with an increase in voltage from 90% (control) to 65% (500V/10 ms). At optimal electroporation parameters (300V/10ms), the number of GFP+/viable cells was 80%/87%. Under optimal transfection conditions, according to digital droplet PCR, the frequency of the targeted knockout after electroporation of TALEN mRNA was 42-49%. According to the SN assay, the frequency of the target knockout after electroporation of Cas9 mRNA/gRNA was 33-39%. Under optimal transfection conditions, no significant change in the colony forming ability of HSCs in the studied samples was revealed.


We have optimized transfection protocol for Cas9/gRNA and TALEN mRNA in primary human hematopoietic stem cells. The results can be used for further development of genome editing therapeutic approaches.


K. V. Lepik thanks the Russian Foundation for Basic Research, grant No. 19-29-04025mk for the financial support.


Genome editing, TALEN nuclease, CRISP/CAS9, hematopoietic stem cells.

Volume 9, Number 3

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doi 10.18620/ctt-1866-8836-2020-9-3-1-152

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