Adenine base editing efficiently restores the function of Fanconi anemia hematopoietic stem and progenitor cells

Siegner SM, Ugalde L, Clemens A, Garcia-Garcia L, Bueren JA, Rio P, Karasu ME, Corn JE.
Source: Nat Commun.
Publication Date: (2022)
Issue: 13(1): 6900
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

Editing LCLs with base editor plasmids

For base editing experiments, LCLs were run through Ficoll gradient and the death cells and debris were cleared. 5 × 10^5 LCLs were electroporated with ABEmax (750 ng) and sgRNA (250 ng) using 4D Nucleofector™ X unit from Lonza (SF solution, DN-100 and CM-137). Cells were cultured in a 24 well dish after nucleofection and transferred into a T25 flask after recovery for the long term culturing.

Editing LCLs with in vitro transcribed mRNA
For base editing with ABEmax mRNA, 2 × 10^5 FA-55 or FA-75 LCLs were electroporated with 3 µg or 6 µg BE mRNA and 100 or 200 pmol of synthetic sgRNAs (Synthego), respectively. For both experiments the Lonza nucleofector was used with SF solution and the EW-113 nucleofection program.

mRNA electroporation

Electroporation was performed using Lonza 4D Nucleofector (V4XP- 3032 for 20-µl Nucleocuvette Strips or V4XP-3024 for 100-µl Nucleocuvette Strips) according to the manufacturer’s instructions. The modified synthetic sgRNA (2'-O-methyl 3' phosphorothioate modifications in the first and last three nucleotides) were purchased from Synthego and BE mRNA was obtained through in vitro transcription using mMESSAGE mMACHINETM T3 Transcription kit (Invitrogen). A total of 2 × 10^5 HSPCs from healthy donor were resuspended in 20 µL P3 solution and electroporated in 20-µL Nucleocuvette wells using program EO-100 with increasing concentration of BE mRNA and sgRNA (3 µg of BEmRNA and 3.2µg sgRNA for HD CB cells and 6 µg of BE mRNA and 6.4 µg sgRNA for HD mPB cells). For 100-µL cuvette electroporation, 1 × 10^6 HSPCs were resuspended in 100 µL P3 solution and electroporated using 30 µg of BE mRNA and 32 µg of sgRNA with program EO-100. FA Lineage negative cells were electroporated using similar conditions. Electroporated cells were resuspended in StemSpan medium (StemCell Technologies) with corresponding cytokines. 


Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused by deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient hematopoietic stem and progenitor cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions leads to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from FA patients was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.