Figure 5.

CRISPR mutants lack catalytically‐active Mmp‐9. (a) Genomic structure of mmp‐9 and the gRNA target sequence. (b) Sequence alignment for two indel mutations—8 bp insertion (mmp‐9 ^mi5004) and 23 bp deletion (mmp‐9 ^mi5003). Red underline indicates the sequence targeted by the 19 bp gRNA. (c) Western blot of retinal proteins from unlesioned retinas (UL wild‐type; UL mmp‐9 ^mi5004; UL mmp‐9 ^mi5003) and lesioned retinas (wild‐type; mmp‐9 ^mi5004; mmp‐9 ^mi5003) at 24, and 48 hpl stained with anti‐Mmp‐9 and anti‐Actin (loading control) antibodies. (d) Zymogram of Mmp‐9 catalytic activity. Lanes in zymogram correspond to those in the Western blot. Purified human recombinant protein serves as a positive control [Color figure can be viewed at wileyonlinelibrary.com]