Genome editing by CRISPR/Cas9 in sorghum through biolistic bombardment

Abstract

The advanced biotechnology CRISPR/Cas9 as a powerful genome editing tool has displayed great potential for improving important agronomic traits such as yield and quality. It has gained momentum worldwide for gene function research of plants in recent years. As for cereals, numerous studies of CRISPR/Cas9 have been reported predominately on rice and quite a few on other cereals including maize, wheat, and barley. In contrast, there are only a couple of reports on sorghum up to date. In this chapter, the CRISPR/Cas9 system has been investigated for sorghum genome editing through biolistic bombardment. Two target genes, cinnamyl alcohol dehydrogenase (CAD) and phytoene desaturase (PDS), have been investigated by CRISPR/Cas9 though bomboarment. Successful genome editing has been achieved within the sorghum genotype Tx430. Furthermore, sequencing PCR product of transgenic plants has confirmed that the CRISPR/Cas9 successfully edited the target gene in sorghum. Both homozygosis and heterozygosis editings of CAD gene have been confirmed in T0 primary transgenic lines through sequencing PCR products. T1 generation of CRISPR plants has been investigated as well. The results illustrated that the edited gene has passed down to next generation. More experiments, such as optimizing promoters for guide RNA (gRNA) and Cas9 in sorghum, are under investigation. Three factors were considered crucial elements to establish an efficient CRISPR/Cas9 system for genome editing in sorghum: (1) an efficient transformation system, (2) the design of targeted gene sequence for gRNA, (3) effective expression of CRISPR components including Cas9 and gRNA.