Figure 1.

CRISPR/Cas9-Mediated Gene Insertion in Mouse Cas9 Transgenic HSPCs

(A) Targeting strategy to insert T2A-mCherry and T2A-BFP into the Lmnb1 and Actb loci, respectively.

(B) Experimental scheme of CRISPR/Cas9-mediated gene insertion in mouse HSPCs.

(C) FACS analysis of the targeted LSK cells 3 days after targeting in vitro. Gates show the percentages of mCherry⁺ or BFP⁺ LSK cells, based on the indicated gating strategy (top left). Circles, squares, and triangles represent individual mice (n = 6) from three independent experiments. Data are shown as means ± SD (^∗∗p < 0.01, Mann-Whitney test).

(D) HR and wild-type (WT) and/or NHEJ fragments were amplified by PCR, using the primers indicated in (A), in control or targeted LSK cells.

(E) Frequencies of HR and NHEJ events in targeted HSPCs that received either sgRNA only or sgRNA and AAV-DJ donor vectors for the Lmnb1 loci (top) and Actb loci (bottom).

(F) Pie charts summarizing the frequency of monoallelic and bi-allelic HR events in the targeted Lmnb1 loci (top) and Actb loci (bottom) of individual reporter⁺ colonies (see also Figure S2B). Data are based on at least three independent experiments. KI, knockin.