Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells.

DeWitt MA, Magis W, Bray NL, Wang T, Berman JR, Urbinati F, Heo SJ, Mitros T, Muñoz DP, Boffelli D, Kohn DB, Walters MC, Carroll D, Martin DI, Corn JE.
Source: Science
Publication Date: (2016)
Issue: 8 (360): 1-9
Research Area:
Stem Cells
Gene Expression
Cells used in publication:
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
4D-Nucleofector™ X-Unit
For each electroporation,150,000-200,000 late log-phase K562 cells were pelleted (100 x g, 5 minutes) and re-suspended in 20 µL Lonza SF solution. 20 µL cells, 10 µL Cas9 RNP containing the desired guide (see above, and Supplementary Table 1), and 1 µL 100 µM ssDNA template programming the desired edit at the SCD SNP were mixed and electroporated using the recommended protocol for K562 cells. After electroporation, K562 cells were incubated for 10 minutes in the cuvette, transferred to 1 mL of K562 media, and cultured for 48-72 h prior to genomic DNA extraction and genotyping. CD34+: To edit HSCs, ~1 million HSPCs were thawed and cultured in StemSpan SFEM medium supplemented with StemSpan CC110 cocktail (StemCell Technologies) for 24 h prior to electroporation with Cas9 RNP. To electroporate HSPCs, 100,000-200,000 were pelleted (200 x g, 10 minutes) and resuspended in 20 µL Lonza P3 solution, and mixed with 10 µL Cas9 RNP and 1 µL 100 µM ssDNA template programming the desired edit. This mixture was electroporated using the Lonza 4d nucleofector and either of two protocols (“1” : DO100, “2”: ER100). Electroporated cells were recovered in the cuvette with 200 µL StemSpan SFEM/CC110 for 10-15 minutes, and transferred to culture in 1 mL StemSpan SFEM/CC110 for 48 hours post-electroporation.
Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the ß-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.