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. Author manuscript; available in PMC: 2023 Apr 22.
Published in final edited form as: Nat Biotechnol. 2022 Aug 25;41(4):521–531. doi: 10.1038/s41587-022-01418-8

Extended Data Fig. 2 |. Comparison of CTS template designs.

Extended Data Fig. 2 |

(a) Diagram of CD5-HA knock-in strategy and control ssDNA HDRTs. (b) Representative flow cytometry plots demonstrating CD5-HA knock-in. (c) Live cell counts for each ssCTS design using a CD5-HA knock-in construct at 160 nM – 4uM concentration. (d-e) Comparison of knock-in efficiency, live cell counts, and knockin cell counts with insertions of increasing size using (d) dsCTS or (e) ssCTS HDRTs targeting the IL2RA gene. Evaluated transgenes encode either GFP (~1.4 kb total HDRT length), an IL2RA-GFP fusion (~2.3 kb total HDRT length), or an IL2RA-GFP fusion plus separate EF1a-mCherry expression cassette (~3.5 kb total HDRT length). Each experiment was performed with T cells from 2 independent healthy human blood donors represented by individual dots + mean. RNP = Ribonucleoprotein, CTS = Cas9 Target Site, ssCTS = ssDNA, HDRT + CTS sites, HDRT = homology-directed-repair template.