苹果愈伤组织CRISPR/LbCpf1基因编辑体系的建立

Establishment of CRISPR/LbCpf1 gene editing system in apple callus

为在苹果愈伤组织中建立CRISPR/Cpf1基因编辑体系,构建双靶点CRISPR/LbCpf1载体,靶向正调控花青苷积累的苹果MdMYB1转录因子基因。转化王林愈伤组织,通过测序以及花青苷积累实验验证敲除效率。结果表明,CRISPR/LbCpf1在苹果愈伤组织中可实现44.4%的总突变率,其中靶点1突变率为22.2%,靶点2为33.3%。CRISPR/LbCpf1易造成2个碱基对(bp)以及7~9 bp的小片段删除,占比68.8%,在距PAM序列远端14~22 bp位置发生突变概率较高,符合典型CRISPR/LbCpf1体系的特点。相对于野生型,成功基因编辑的愈伤组织株系花青苷积累明显减少,综上说明CRISPR/LbCpf1体系可在苹果愈伤组织中实现较高效的基因编辑。

CRISPR/Cpf1 is a series of gene editing systems with high mutation efficiency and easier building of multiple gene-editing elements. To establish the CRISPR/Cpf1 system in apple callus, the dual-target CRISPR/LbCpf1 vector was constructed to target the MdMYB1 transcription factor gene that positively regulates anthocyanin accumulation. After transformation of callus, the knockdown efficiency was verified of by sequencing and anthocyanin accumulation experiments. Based on the results, CRISPR/LbCpf1 achieved a total mutation rate of 44.4% in apple callus, with a mutation rate of 22.2% in target 1 and 33.3% in target 2. Mutation types analysis showed that CRISPR/LbCpf1 tended to cause deletion of 2 base pairs(bp) and small fragments of 7~9 bp, accounting for 68.8%, and the probability of mutation at 14~22 bp distal to the PAM sequence was high, which was consistent with the characteristics of typical CRISPR/LbCpf1 system. The anthocyanin accumulation in the successfully gene-edited callus lines was significantly reduced compared with the wild type. In summary, the CRISPR/LbCpf1 system could achieve higher efficient gene editing in apple callus.