Fig. 1

Introduction of the R172P substitution in TRP53 locus via a single injection of the CRISPR/Cas9 system. a Procedure of construction of KI mice: Step 1) Design gRNA and donor for mouse engineering; Step 2) In vitro transcription to generate mRNA of Cas9 and gRNA; Step 3) Prepare zygotes for microinjection and inject Cas9 mRNA, gRNA and donor into zygotes as well as transplant obtained zygotes into foster mother mice. b A schematic illustration shows the designation of gRNA and donor in TRP53 R172P KI mouse engineering. The gRNA closest to the mutation site was selected where the mutation site(g- > C) locates5 nucleotides downstream of gRNA NGG (PAM site). A 120-nucleotide length oligo donor was used, which covered the gRNA and mutation site (top panel). The donor carried 6 silent mutations, which do not cause amino acid replacement, in the gRNA region and a G- > C mutation to generate the R172P substitution of the TRP53 tumour suppressor (bottom panel). c The PCR product contained a T7 promoter before the hSpCas9 cDNA with an approximate length of 4300 bp (top panel). PCR amplification of hSpCas9 cDNA from the pX260 vector was performed using the Phusion high-fidelity PCR kit, and the single band product with the correct length is shown in the agarose gel (left panel). The agarose gel image (right panel) shows that the MEGAclear kit purification and poly A addition products had the same length and were longer than the in vitro transcription product of T7 RNA polymerase. d The PCR product contained a T7 promoter, gRNA and tracrRNA with an approximate length of 120 bp (top panel). PCR amplification of gRNA DNA with high-fidelity PCR kit resulted in a single band product with the correct length as shown in the agarose gel (left panel). The in vitro transcription product of gRNA was validated by agarose gel electrophoresis (right panel)