A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing

Abstract

CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing.

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

“…CLA1 encodes 1-deoxy-D-xylulose-5-P synthase (Mandel et al, 1996;Estévez et al, 2001;Tang et al, 2010) and IspH is for an isopentenyl/dimethylallyl diphosphate synthase involved in isoprenoid biosynthesis. Their null mutations or VIGS resulted in albino phenotypes (Rohdich et al, 2002;Hsieh and Goodman, 2005;Yin et al, 2015). 60-bp invertedrepeat fragments targeting SiPDS, SiCLA1, and SiIspH were inserted into pFoMV-sg to generate FoMV-SiPDS, FoMV-SiCLA1, and FoMV-SiIspH, respectively.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…Indeed, biolistic co-transformation in rice with plasmids bearing a donor cassette released in planta gave higher gene knock-in efficiency with additional free dsDNA donor fragments (36% of lines regenerated on selective media) than without (25% of lines regenerated on selective media) [67]. To strongly increase their copy number in the transformed cells, the sequences coding for the CRISPR-Cas9 system and the donor template can also be inserted into disarmed viral replicons of geminiviruses [8,27,89,90]. Other types of viral vectors, such as Tobacco Mosaic Virus or Tobacco Rattle Virus, have been long used for transient expression of recombinant proteins in plants [91], but these viral constructs are too low cargo for the delivery of the CRISPR coding elements all together.…”

Recent Biotechnological Advances in the Improvement of Cassava

“…However, only a few reports of use of CRISPR/Cas9 system in cassava improvement exist and are still in the preliminary stage, these include CRISPR/ Cas9-mediated modification of cassava flowering genes to induce flowering in this predominantly clonally propagated crop [97]. Because of the successful development of a modified geminivirus vector based on Cabbage leaf curl virus for a virus-guided delivery of CRISPR/Cas9 [98], it is likely that a similar vector system can be developed for cassava using the cassava geminivirus, African cassava mosaic virus.…”