CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool
(A) In vitro cleavage depending on sgRNA-1/Cas9 occurred on linearized plasmids containing eGFP but not eGFPvar. Successful cleavage of eGFP plasmid (4128bp) resulted in a 2052bp and 2076bp fragment. The absence of expected fragments (627bp, 4025bp) demonstrated that eGFPvar (4652bp) was not digested by sgRNA-1/Cas9. (B) A faint double band (2154bp, 2198bp, asterisk) indicated inefficient digestion of off-target 1 (OT#1) while OT#2 and OT#3 (S1 Table) were not cleaved. Note: contrast was enhanced for better visualization. (C) Silent mutations of sgRNA-1 target site in eGFPvar. (D-E) Injections of sgRNA-1 and Cas9 mRNA into wimb-/+ embryos (D) resulted in strong inactivation of eGFP (E).