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. 2019 Jan 1;6:7. doi: 10.1038/s41438-018-0082-6

Fig. 2. Overview of single target site or multiple sgRNA expression cassette assembly.

Fig. 2

a Cloning of single target site in the pHNCas9HT and transformation in Agrobacterium competent cells. pHNCas9HT were digested by Esp3 I and purified. b Preparation of mixture of templates for AtU3b-sgRNA, AtU3d-sgRNA, AtU6-1-sgRNA, AtU6-26-sgRNA, and AtU6-29-sgRNA (all at 1 ng/µl). AtU-F is a universal primer for the five AtU promoters. AtUX designates specific primers for AtU3b-R, AtU3d-R, AtU6-1-R, AtU6-26-R, and AtU6-29-R, respectively. c The sgRNA expression cassettes with target site were generated by one-step PCR in one pot. d Assemble multiple sgRNA expression cassettes in pHNCas9. The pHNCas9 was digested by Esp3 I and purified