Journal: bioRxiv

Article Title: Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells

doi: 10.1101/2023.05.27.542323

Figure Lengend Snippet: Efficient editing and HbF induction by targeting the +55 BCL11A erythroid enhancer in CD34+ HSPCs (A) Fifteen sgRNAs were designed to target core +55 BCL11A enhancer. Half E-Box/GATA (TGN7-9WGATAR) motifs which are binding sites for erythroid transcription factors TAL1 and GATA1 marked in the frame. (B) Indel frequency of 3xNLS-SpCas9 complexed with various gRNAs in CD34+ HSPCs measured by TIDE analysis. Data are plotted as mean ± SEM, n = 3 healthy donors (HD 4, HD 5 and HD6). (C) HbF induction by HPLC analysis in erythroid cells in vitro differentiated from RNP edited CD34+ HSPCs from three healthy donors (HD 4, HD 5 and HD 6). Data are plotted as mean ± SEM and analyzed with the unpaired two-tailed Student’s t -test. ns: nonsignificant, **** P < 0.0001. n = 3 healthy donors. (D) Indels analyzed by CRISPResso 2 following 3xNLS-Cas9: sg1449 RNP electroporation of CD34+ HSPCs. (E) Indels analyzed by CRISPResso 2 following 3xNLS-Cas9: sg1450 RNP electroporation of CD34+ HSPCs. (F) Reduction of BCL11A mRNA expression compared to safe locus editing by ddPCR in erythroid precursors. Data are plotted as mean ± SEM, n = 3 healthy donors (HD 1, HD 2 and HD 3). (G) Cell expansion during erythroid differentiation. Data are plotted as mean ± SEM, n = 3 healthy donors (HD 1, HD 2 and HD 3). (H) Enucleation by Hoechst staining at the terminal erythroid differentiation. Data are plotted as mean ± SEM, n = 3 healthy donors (HD 1, HD 2 and HD 3). (I) Fraction of F cells by flow cytometry analysis. Data are plotted as mean ± SEM and analyzed with one-way ANOVA. ns: nonsignificant. n = 3 healthy donors (HD 1, HD 2 and HD 3). (J) HbF levels by HPLC in in vitro differentiated erythroid cells from three healthy donors (HD 1, HD 2 and HD 3). Data are plotted as mean ± SEM and analyzed with one-way ANOVA. ns: nonsignificant, *** P < 0.001.

Article Snippet: For Lonza 4D large scale electroporation, 1500 pmol (15 μM) of sg1617 or sg1618 was mixed with 500 pmol (5 μM) of 3xNLS-SpCas9-SpCas9 protein and added electroporation buffer (Lonza 4D, cat# V4XP-3024) up to 25 μl in one tube, 1500 pmol (15 μM) of sg1450 or sg1449 was mixed with 500 pmol (5 μM) of 3xNLS-SpCas9 protein and added electroporation buffer up to 25 μl in another tube.

Techniques: Binding Assay, In Vitro, Two Tailed Test, Electroporation, Expressing, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells

doi: 10.1101/2023.05.27.542323

Figure Lengend Snippet: Gene editing HSPCs without ex vivo culture evades genotoxicity. (A) Cell size by forward scatter without (0 h) and with 24 h and 48 h of cytokine culture. (B) Fractions of cells in G0, G1, S and G2/M immediately after thawing or selection, 24 h and 48 h of pre-stimulation culture from three cryopreserved healthy donors mobilized with G-CSF and one fresh SCD patient donor mobilized with plerixafor (HD 2, HD 3, HD 31 and SCD 3) . Data are plotted as mean ± SEM. (C) Micronucleus analysis by In Vitro Micro Kit 48 h after RNP electroporation in CD34+ HSPCs without and with 24 h and 48 h of cytokines culture. Data are plotted as mean ± SEM and analyzed with one-way ANOVA. ns: nonsignificant, * P < 0.5, ** P < 0.01. n = 3-5 healthy donors (HD 2, HD3 and HD 32-34). (D) Design of ddPCR assays recognizing chr2p (telomeric to the BCL11A cleavage site) and chr1 as a reference autosome. (E) Enrichment of chr2p in sorted nuclei by ddPCR assay. Data are plotted as mean ± SEM and analyzed with one-way ANOVA. ns: nonsignificant. n = 3-5 healthy donors (HD 2, HD3 and HD 32-34). (F) Enrichment of chr2p in sorted micronuclei by ddPCR assay. Data are plotted as mean ± SEM and analyzed with one-way ANOVA. ns: nonsignificant, * P < 0.5, ** P < 0.01. n = 3-4 healthy donors (HD 2, HD3 and HD 32-34). (G) Overall editing by +58 drop-off ddPCR assay. Data are plotted as mean ± SEM and analyzed with one-way ANOVA. ns: nonsignificant, * P < 0.5, ** P < 0.01. n = 3-5 healthy donors (HD 2, HD3 and HD 32-34). (H) Frequency of long deletions/rearrangements (not including the templated 3 kb deletion/inversion expected for double editing) was calculated as overall edit - deletion - inversion - small indels from four ddPCR assays. Left was measured by +58 drop-off and +58 offset assays. Right was measured by +55 drop-off and +55 offset assays. Data are plotted as mean ± SEM and analyzed with one-way ANOVA. ns: nonsignificant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n = 3-5 healthy donors (HD 2, HD3 and HD 32-34). (I) Frequency of long deletions/rearrangements (not including the templated 3 kb deletion/inversion expected for double editing) in engrafting cells as compared to input cells. Data are plotted as median with range. n = 3 donors for input (SCD 3, HD 29 and HD 35 for sg1617+sg1450 and sg1618+sg1449. HD 7, HD 13 and HD 14 for sg1618+sg1450), n = 9-12 primary recipients for engrafted. (J) CDKN1A expression, by RT-qPCR, in CD34+ HSPCs 5 (left) and 24 (right) hours after RNP electroporation from three healthy donors. Relative expression normalized to control sample 5 hours of pre-stimulation. Data are analyzed with the unpaired two-tailed Student’s t -test. ns: nonsignificant. * P < 0.05. n = 3 healthy donors (HD 1, HD 2 and HD 3).

Article Snippet: For Lonza 4D large scale electroporation, 1500 pmol (15 μM) of sg1617 or sg1618 was mixed with 500 pmol (5 μM) of 3xNLS-SpCas9-SpCas9 protein and added electroporation buffer (Lonza 4D, cat# V4XP-3024) up to 25 μl in one tube, 1500 pmol (15 μM) of sg1450 or sg1449 was mixed with 500 pmol (5 μM) of 3xNLS-SpCas9 protein and added electroporation buffer up to 25 μl in another tube.

Techniques: Ex Vivo, Selection, In Vitro, Electroporation, Expressing, Quantitative RT-PCR, Two Tailed Test