Figure 5.

ArrayEdit Rapidly Produces Living, Well-Characterized, and Functional LAMA5 Gene-Edited hESC Lines

(A) Representative images of hESC colony formation of each biallelic edited line on both matrigel- and laminin111-coated substrates. Laminin-111 contains less matrix proteins thereby accentuating self-renewal defects generated from loss of α-5 laminin protein domains in the biallelic edited cells. Clone G10 notably displayed a drastic decrease in cell number on laminin-111. WT, wild-type.

(B) Detection of apoptosis levels by immunocytochemistry of cleaved caspase-3. Each biallelic clone had detectable apoptotic cells (some are denoted by arrowheads). All scale bars represent 100 μm.

(C and D) Mean number of cells (C) and cleaved caspase-3 positive cells (D) ±1 SD for each hESC clone on matrigel, laminin-111, laminin-521 coated substrates after 4 days in culture on various substrates as determined by flow cytometry (n = 4; independent experiments). On matrigel and laminin-111 substrates, each biallelic edited line was less dense than wild-type cells (Student's two-tailed t test, p < 0.05), indicating a functional defect in self-renewal. On laminin-521 substrates, each clone was at least as dense as wild-type cells, and had a similar number of apoptotic cells as wild-type except for G10. This may be due to complex deletions in the G10 clone. Clone G10 could not be detected (n.d.) due to a low cell number on laminin-111. Clone C8 had an in-frame mutation that may be partially rescued on the matrigel substrate and had a significantly higher number of cells on laminin-521.

(E) Comparison of ArrayEdit against standard methods to produce gene-edited cells. ArrayEdit is approximately 2× faster and produces significantly more gene-edited cell lines (see Figure 4D). ArrayEdit reduces or eliminates several steps in the gene-editing workflow and additionally has important multiplexing and HCA capabilities. See also Figure S5.