*2.1. Knockout of OsCAF1 Produced the Albino Seedling Phenotype*

According to a previous study, the ortholog of *ZmCAF1* in rice was *Os01g0495900*, which was named *OsCAF1*, and the function of OsCAF1 has not been determined to date [8]. We observed that the cDNA of *OsCAF1* had a 2106-bp nucleotide and encoded 701 amino acids (aa). To determine the function of OsCAF1 in rice chloroplast development, *oscaf1* mutants were generated using the CRISPR/Cas9 system (Figure 1A,B), and 26 transgenic plants were obtained in the T0 generation. Sequencing and phenotypic analysis revealed that *oscaf1* homozygous mutants exhibited the albino phenotype in the rice seeding stage (Figure 1C,D), and then died after the three leaves stage. In both of #3 and #4 mutants, a premature stop codon was created by a frame shift in the coding region of *OsCAF1*. However, the *OsCAF1*/*oscaf1* heterozygous exhibited green leaves (Figure 1C,D), which exhibited normal growth and phenotypes under chamber conditions. Because of *oscaf1*/*oscaf1* homozygous lethal mutants, the heterozygous lines were used to generate the T1 generation. The different homozygous mutations in the allele's of *OsCAF1* in T1 generation were obtained from #1 and #2 plants and also exhibited albino seedling phenotypes. These results indicated that *OsCAF1* mutations caused the albino seedling phenotype. To study the function of OsCAF1, *oscaf1* mutants from T1 generation of #1 plant were selected for further analysis.


**Figure 1.** Knockout of *OsCAF1* produces the albino phenotype in seedlings. (**A**) Diagram of CRISPR/ Cas9 system for editing OsCAF1. (**B**) Schematic diagram of the targeted site in OsCAF1. (**C**) Mutation types of four positive plants in T0 generation. The targeted sequences are in blue letters. The protospacer adjacent motif (PAM) sequences are in red letters. The number of nucleotides at both sides of the target sequences is 18 bp. (**D**) Phenotypes of four positive plants at seedling stage. Scale bar, 1 cm.
