Fig. 5
From: Correction to: A Versatile Vector Toolkit for Functional Analysis of Rice Genes
Diagram of how CRISPR/Cas9 vectors were constructed and used to edit the rice IPA1 gene. a Western blot analysis of the Cas9-intron expression in rice protoplasts. HSP indicates the loading amount of each sample. b Target site selection for candidate genes in the rice genome. A 20-bp specific sequence followed by the PAM “NGG” structure is required. c and d Target cloning to the entry vectors. Synthesis of the primer pairs of the 20-bp specific target with the 4-bp adapters, and ligation with the BsaI linearized pEntry A or pEntry B vector. e One-step ligation and f step-by-step ligation of multiple targets to pRHCas9/pRGCas9. Four pairs of isocaudamers, PstI(E1)-NsiI(E1'), XbaI(E2)-SpeI(E2'), BamHI(E3)-BglII(E3'), and SalI(E4)-XhoI(E4') are marked. The sgRNA cassettes with U6P1 and U6P2 in pEntry A and B should be used in turn. g Representative sequencing chromatogram of the CRISPR-IPA1 transgenic lines. Line #7, wild-type genotype; line #5, mutant genotype. h Representative gene editing results in the CRISPR-IPA1 transgenic lines