Abstract: A highly efficient and safe gene site-editing system was established to obtain Cas9-Free Arabidopsis ALB3 homozygous mutant seedlings,providing ideal materials for studying photosynthesis pathways and physiological metabolic processes of plants. Designed two sgRNAs for the Arabidopsis thaliana target gene ALB3, the Cas9 protein was induced to recognize the PAM site, thereby realized large fragment knockdown of the ALB3 gene;Assembled fluorescent screening marker gene mCherry into CRISPR/Cas9 vector to construct a CRISPR/Cas9 vector with fluorescent screening marker;The Agrobacterium transformation method was used to transform Arabidopsis thaliana, and its offspring were screened and verified. The homozygous mutant plants of Cas9-Free were obtained by selecting under an inverted fluorescence microscope.The sequencing results showed that the sequence of A.thaliana ALB3 editing vector containing the visual screening marker gene was consistent with the expectation,thus demonstrated the vector was successfully constructed; observed by inverted fluorescence microscope,positively transformed Arabidopsis T₀ seeds contain fluorescent markers. Apparent observation and molecular identification indicated that the T₁ generation plants obtained homozygous mutant progeny,strip size was smaller than wild type,and the whitening mutation traits were obvious. The T₂ generation plants grown from T₁ generation non-fluorescent seeds had been successfully implemented Cas9-Free and the plants were bleached. This study successfully constructed a CRISPR/Cas9 vector with a fluorescent selection marker and realized large fragment knockdown of the Arabidopsis ALB3,obtained a homozygous mutant of Cas9-Free,provided reference for the construction of A.thaliana gene efficient editing vector and the acquisition of Cas9-Free gene editing progeny.

Key words: CRISPR/Cas9, ALB3 gene, Arabidopsis thaliana, Gene editing, Vector

CLC Number: 

• Q782

• Q786

• Q789