3/1/2023 0 Comments Snapgene lenticrispr v2 map![]() Primer targeting Cas9 is specific for the gene editors while that targeting U6 promoter is specific for gRNA primer targeting WPRE is common for both gRNA and gene editors. ( a) Vector copy number (VCN) per cell for the integrated guide RNA (gRNA) as well as the gene editors (ABE, CBE, and Cas9) in the respective HUDEP-2 stable cell lines. Data are expressed as mean ± SEM from three biological replicates, asterisks indicate levels of statistical significance (**p < 0.01). The data were normalized with respective controls. ( d) Expression of G gamma-globin chain in ABE, CBE, and Cas9 edited HUDEP-2 cells, measured by RP-HPLC after differentiation into erythroblasts. ( c) Analysis of HBG2 deletion (due to 4.9 kb deletion) by qRT-PCR in the base edited and Cas9 edited HUDEP-2 cells. The percentage of HbF-expressing cells were analyzed before and after differentiation into erythroblasts. ( b) Flow cytometry analysis of fetal hemoglobin (HbF) and erythroid maturation markers (CD235a and CD71) expression in edited HUDEP-2 cells. The transduction efficiency was analyzed by FACS, the individual base substitution and indel percentage were analyzed by EditR and ICE software respectively after sanger sequencing. ( a) Transduction efficiency of gRNA-2 (for ABE and CBE) or gRNA-7 (for Cas9), percentage of individual base conversion for ABE and CBE (with gRNA-2) and insertions and deletions (indels) for Cas9 (with gRNA-7) before and after erythroid differentiation are represented. Highly homologous HBG promoter was edited by adenine base editor (ABE), cytosine base editor (CBE), and Cas9 with suitable guide RNAs (gRNAs) that target the well-known BCL11A binding site (–115 transcription start site ). Institute of Molecular Health Sciences, Department of Biology, Switzerland.Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, United States.Department of Biochemistry, Christian Medical College, India.Research and Development Department, Central Blood Institute Blood Service Headquarters, Japanese Red Cross Society, Japan, Japan.Cell Engineering Division, RIKEN BioResource Center, Japan.Department of Haematology, Christian Medical College & Hospital, India.Manipal Academy of Higher Education, India.School of Biotechnology and Biomolecular Sciences, University of New South Wales, Australia.Institute of Data Science and Biotechnology, Gladstone Institutes, United States.Innovative Genomics Institute, University of California, Berkeley, United States.Sree Chitra Tirunal Institute for Medical Sciences and Technology, India.Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, India. ![]()
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