Journal: bioRxiv

Article Title: Efficient Methods for Target Gene Manipulation in Haematopoietic Stem Cell Derived Human Neutrophils

doi: 10.1101/2023.06.17.545406

Figure Lengend Snippet: A Map of the payload for U6-shRNA vectors. This is a bicistronic construct con-taining U6-shRNA followed by PGK-Puro-T2A-BFP. B Secondary structure of U6-driven shRNA hairpin. The predicted transcript contains a 21nt dsRNA stretch separated by a 9nt hairpin derived from hsa-miR21. C CD11b expression assessed at day 20 of differentiation by flow cytometry. Cells underwent staggered transduction during differentiation with lentivirus carrying three different U6-driven shRNA’s targeting ITGAM, as well as a nontargetign control. D Map of the payload for the tagBFP-miRshRNA vector, with the shRNA in the 3’UTR of tagBFP. In all maps, lentiviral elements up-and downstream of the payload are not shown. E Secondary structures of endogenous hsa-miR-30a (left) and miR30a-ITGAM-sh5 which contains a guide sequence targeted to the 3’LTR of ITGAM . The structure of the synthetic shRNA differs from endogenous in missing two bulges: a 1nt mismatch at position 12, and a 2nt mismatch at positions 30-32. All structures were generated with the RNAfold Webtool . F Experiments showing CD11b expression by flow cytometry at day 20 of differ-entiation after staggered transduction with lentivirus carrying miR-shRNA’s. Five different miR-shRNA’s targeted to ITGAM as well as a nontargeting control were tested. Data for both tagBFP + cells (top) or all live single cells (bottom) are shown. UT, untreated; NT, nontargeting shRNA; ITGsh1-5, shRNA’s 1-5 targeted to ITGAM . For and , individual data from six distinct donors are shown as dots, with horizontal bars indicating the median of each group with the interquartile range depicted by error bars. Asterisks indi-cate statistical significance (Friedman’s test) as detailed by bars between relevant groups: *p ≤ 0.05;**p ≤ 0.01; ***p ≤ 0.001. Gating strategy for all flow cytometry is shown in Fig. S1C, and was obtained on the Attune NxT flow cytometer.

Article Snippet: The following antibodies or staining reagents were used: Mouse-anti human CD11b-FITC (Bear-1, Hycult Biotech), mouse-anti human CD62L-Alexa Fluor 647 (DREG-56, Biolegend), mouse-anti human CD16-Pacific blue (3G8, Biolegend), mouse anti-human CD11b-Alexa Fluor 647 (ICRF44, Biolegend), Annexin V-FITC (Thermofisher Scientific), propidium iodide (Biolegend).

Techniques: shRNA, Construct, Derivative Assay, Expressing, Flow Cytometry, Transduction, Control, Plasmid Preparation, Sequencing, Generated

Journal: bioRxiv

Article Title: Efficient Methods for Target Gene Manipulation in Haematopoietic Stem Cell Derived Human Neutrophils

doi: 10.1101/2023.06.17.545406

Figure Lengend Snippet: A Differentiation timeline with nucleofection (red) after 3 days of StemSpan preconditioning. Nucleofected cells were rested in media containing StemSpan and 5 µ M Z-VAD-FMK, a pan-caspase inhibitor, to mitigate cell losses after nucleofection. Cells were then differentiated G-CSF at 10ng/mL for 14 days. B,C CD11b expression by flow cytom-etry at day 20. MFI (B), and total CD11b - the product of percent positive and MFI of the positive cells (C) are shown. D Genomic indel profiles obtained by Sanger sequencing and Tracking of Indels by DEcomposition (TIDE) analysis comparing ITGAM guide RNA 1 compared to donor-specific nontargeting controls. The Cas9 cut site is shown as 0nt, and insertions (right, positive) and deletions (left, negative) are expressed as a percentage of total sequences. Data at 0nt are indicate no sequence change. Total efficacy is shown at the left of the graph and is the sum of percentage indels. E,F Functional capacities of CRISPR-edited neutrophils. Phagocytosis of serum opsonised pHrodo red conjugated bioparticles of S. aureus (SA) and E. coli (EC) are shown in (E), and oxidative response by DHR fluores-cence are shown in (F). NT, Nontargeting guide; ITGAM KO, ITGAM gRNA1. For and 5E-F, individual data from six distinct donors are shown as dots, with horizontal bars indicating the median of each group with the interquartile range depicted by error bars. Asterisks indicate statistical significance (Friedman’s test) as detailed by bars between rel-evant groups: *p ≤ 0.05;**p ≤ 0.01; ***p ≤ 0.001. Gating strategies are shown in Fig. S1D and all flow data was obtained on the Attune NxT flow cytometer. Example outputs of TIDE analysis are shown in Fig.S2.

Article Snippet: The following antibodies or staining reagents were used: Mouse-anti human CD11b-FITC (Bear-1, Hycult Biotech), mouse-anti human CD62L-Alexa Fluor 647 (DREG-56, Biolegend), mouse-anti human CD16-Pacific blue (3G8, Biolegend), mouse anti-human CD11b-Alexa Fluor 647 (ICRF44, Biolegend), Annexin V-FITC (Thermofisher Scientific), propidium iodide (Biolegend).

Techniques: Expressing, Sequencing, Functional Assay, CRISPR, Flow Cytometry