Extended Data Fig. 3: Super-adaptation in Δtracr is not due to lack of interference or changes to cas expressions.

a, Schematic of Nme strains used in Fig. 2b, c and Extended Data Fig. 3b–h. b, Northern blot confirming tracrRNA’s absence and complementation. Total RNAs were probed for tracrRNA (top), crRNA (bottom), and 5S rRNA (loading control, middle). Mature crRNAs disappeared in Δtracr as expected, as they must co-load into Cas9 along with tracrRNA to stably exist. c, MDAΦ versus host breakdown for DNA source of new spacers from “+1” bands of lanes 2 and 4 in Fig. 2b. In super-adaptation, viral DNA remained preferred over the host genome. Red dashed line, theoretical random breakdown without any viral vs. self- discrimination. Data are mean ± s.d., n = 3. d-e, Super-adaptation is not due to changes in Cas9 or Cas1 protein levels. Anti-Cas9 (d) and anti-Cas1 (e, w/ or w/o IPTG induction) western blots showed comparable protein levels between Δtracr and WT strains. GroEL, loading control. f, Interference assay for strains used in Fig. 2b. Interference is abolished by Δtracr and restored by tracr complementation. Data are shown as in Extended Data Fig. 2k. g, Strains isogenic to those in Fig. 2b but encode dcas9 instead of WT cas9 showed similar super-adaptation phenotypes. Top, a representative adaptation PCR gel; bottom, quantification of adaptation efficiencies. Data are mean ± s.d., n = 3. NS, not significant (P ≥ 0.05), * 0.005 ≤ P < 0.05, ** P < 0.005; P values calculated by two-tailed Welch’s t-tests. h, 3′ motif analysis for new viral spacers from panel g.