Efficient DIPA-CRISPR mediated knockout of an eye pigment gene in the white-backed planthopper, Sogatella furcifera (Horváth)

Authors: ZHANG, MENG-QI - Guizhou University; GONG, LANG-LANG - Guizhou University; LONG, GUI-JUN - Guizhou University; MA, YUN-FENG - Guizhou University; GUO, HUAN - Guizhou University; LIU, XUAN-ZHENG - Guizhou University; Hull, Joe; DEWER, YOUSSEF - Guizhou University; YANG, CHAO - Guizhou Jifeng Seed Industry Llc; NING-NING, ZHANG - Weifang University Of Science And Technology; HE, MING - Guizhou University; HE, PENG - Guizhou University

Submitted to: Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/13/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary: CRISPR/Cas9-based gene editing is a powerful tool for examining how genes impact insect biology and behavior. The method typically requires microinjection of nanoliter volumes into newly laid insect eggs. However, not all eggs are amenable to the microinjection technique due to size, shell hardness, and/or difficulty in extracting the eggs from the substrate in which they were laid. An alternative approach recently reported for insects is DIPA-CRISPR (direct parental CRISPR), which bypasses direct manipulation of the egg for microinjection of the gene editing cargo into appropriately aged females. The injected cargo is then taken up along with yolk protein precursors by the developing oocyte, thereby avoiding the pitfalls and limitations typically associated with delivering gene editing components into the egg. The utility of this method was evaluated using white-backed planthoppers, which are poor candidates for traditional CRISPR/Cas9 approaches, by injecting females at varying ages post-adult emergence with gene editing cargo targeting an enzyme critical for eye coloration. Edited progeny would thus be distinguishable by red-colored eyes rather than the typical olive coloration. The highest degree of gene editing was observed in females injected two days after adult emergence, confirming the viability of this CRISPR approach. The study demonstrates the utility of DIPA-CRISPR gene editing for insect species that are less amenable to egg-based injections.

Technical Abstract: Although CRISPR/Cas9 has been widely used in insect gene editing, the need to conduct microinjections with preblastoderm embryos can preclude applicability of the technique in insect species with eggs that are small, have hard shells, and/or are difficult to collect and maintain outside of their normal environment. Such is the case with the white-backed planthopper (WBPH), a significant pest of rice that oviposits inside rice stems. Egg extraction from the stem runs the risk of mechanical damage and hatch is heavily influenced by the rice stem micro-environment. To bypass these issues, we targeted embryos prior to oviposition via direct parental (DIPA)-CRISPR in which Cas9 and sgRNAs for the WBPH eye pigment gene tryptophan 2,3-dioxygenase were injected into the hemocoel of adult females. Females at varying days post-eclosion were evaluated to determine at what stage their oocyte might be most capable of taking up the gene editing components. Evaluation of the offspring indicated that the highest G0 gene-edited efficacy (55.4%) occurred in females injected two days post-eclosion and that mutations were heritably transmitted to the G1 generation. This study demonstrates the potential utility of DIPA-CRISPR for future gene editing studies in non-model insect species and can facilitate the development of novel pest management applications.