Extended Data Fig. 2: Plasmid retention assay.
From: Anoxia activates CRISPR–Cas immunity in the mouse intestine
CRISPR-Cas activity was measured by retention frequency of a CRISPR target plasmid. The CRISPR target plasmid encodes a gentamycin resistance (gentR) gene and a protospacer adjacent motif (PAM) followed by a protospacer sequence matching the native CRISPR array. The control plasmid is identical, except that the protospacer sequence is replaced with a protospacer not recognized by the native CRISPR-Cas system. CRISPR-Cas activity converts cells from gentamycin resistant to gentamycin sensitive (gentS) by causing cleavage and loss of the CRISPR target plasmid, but not the control plasmid. To measure CRISPR-Cas activity in culture, gentamycin resistant cells were streaked onto plates and cultured in oxic or anoxic conditions for 24-hours. After culture, single colonies were picked and total versus gentR CFU was determined by serial dilution.
