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. 2016 Jun 23;84(7):1994–2001. doi: 10.1128/IAI.00077-16

FIG 3.

FIG 3

Generation and verification of NTPase I and II single- and double-disruptant mutants. (A) Diagram of single NTPase II (Δntpase II; RHΔhxgprt/ntpaseII::DHFR) and NTPase I (Δntpase I; RHΔhxgprt/ntpaseI::DHFR) mutants and two versions of double-knockout mutants (Δntpase II Δntpase I, RHΔhxgprt/ntpaseII::DHFR/ntpaseI::HXGPRT; Δntpase II Δntpase I v.2, RHΔhxgprt/ntpaseII::HXGPRT/ntpaseI::HXGPRT) generated by CRISPR/Cas9 guided gene disruption. The pseudogene (PG) lacks a suitable start codon and therefore is not transcribed (30). However, it shares complete sequence homology in part with either NTPase II or I for which reason the guide RNA II targets both NTPase II and the pseudogene resulting in a double cut. The type I RH strain (Δhxgprt) was the background for all the mutants described. For the locations and sequences of guides, see Table S3 in the supplemental material. (B) Diagnostic PCR of disruptant mutants with primer pair locations as indicated in panel A. For primer locations and sequences, see Table S3 in the supplemental material. (C) Western blot analysis of protein loss in NTPase disruptant mutants. GRA7 served as loading control.