CRISPR/Cas9 mediated high efficiency knockout of the eye color gene Vermillion in Helicoverpa zea (Boddie)

Abstract

Among various genome editing tools available for functional genomic studies, reagents based on clustered regularly interspersed palindromic repeats (CRISPR) have gained popularity due to ease and versatility. CRISPR reagents consist of ribonucleoprotein (RNP) complexes formed by combining guide RNA (gRNA) that target specific genomics regions and a CRISPR associated nuclease (Cas). The gRNA targeting specific gene sequences may be delivered as a plasmid construct that needs to be transcribed or as a synthetic RNA. The Cas nuclease can be introduced as a plasmid construct, mRNA, or purified protein. The efficiency of target editing is dependent on intrinsic factors specific to each species, the target gene sequence, and the delivery methods of CRISPR gRNA and the Cas nuclease. Although intrinsic factors affecting genome editing may not be altered in most experiments, the delivery method for CRISPR/Cas reagents can be optimized to produce the best results. In this study, the efficiency of genome editing by CRISPR/Cas system in the bollworm, Helicoverpa zea (Boddie), was evaluated using ribonucleoprotein (RNP) complexes assembled by binding synthetic gRNA with purified Cas9 nuclease engineered with nuclear localization signals to target the vermillion (eye color) gene. Mutation rates of adults emerging from embryos microinjected with 1, 2, or 4 μM RNP complexes were compared using replicated experiments. Embryos injected with 2 or 4 μM RNP complexes displayed significantly higher mutation rates (>88%) in surviving adults compared to those injected with 1 μM. The hatch rate in embryos injected with RNP complexes and with injection buffer only (mock injections) was reduced by 19.8(±5.2)% compared to noninjected control embryos, but did not differ significantly between injected embryos. Evaluation of potential off-target sites in H. zea genome did not identify any mutations. This study demonstrates that in vitro assembled synthetic RNP complexes can be used to obtain high genome editing rates in a reproducible manner in functional genomics or genetic manipulation studies.

RNA interference‐mediated knockdown of eye coloration genes in the western tarnished plant bug (Lygus hesperus Knight)

“…Although numerous genes involved in eye coloration have been identified and successfully utilized as transgenic markers in a number of insect species (Coates, Jasinskiene, Miyashiro, & James, ; Cornel et al, ; Fridell & Searles, ; White et al, ; Zwiebel et al, ), the associated eye color phenotypes are not always conserved across species. In D. melanogaster and S. americana , disruption of vermilion results in a red‐eye phenotype (Dong & Friedrich, ; Walker et al, ), whereas in Helicoverpa armigera , Musca domestica , and T. castaneum mutations of the same gene yielded yellow, green, and white eyes (Lorenzen et al, ; Perera et al, ; White et al, ). Similarly, cinnabar mutations manifest as red eyes in D. melanogaster , Leptopilina boulardi , Nasonia vitripennis , and N. lugens (Colinet et al, ; M. Li et al, ; Warren et al, ; Xue et al, ), but are white in T. castaneum , B. mori , and Aedes aegypti (Aryan, Anderson, Myles, & Adelman, ; Lorenzen et al, ; Quan et al, ).…”

“…A number of genetic loci initially found to influence Drosophila eye coloration have since been shown to correspond to genes encoding biosynthetic pathway enzymes, ATP‐binding cassette (ABC) transporters, and vesicular transport proteins (Lloyd et al, ). Given the ease of visual screening, eye color mutations have been used as genetic markers for germ‐line transformation (Cornel, Benedict, Rafferty, Howells, & Collins, ; Sethuraman & O'Brochta, ; White, Coates, Atkinson, & O'Brochta, ; Zwiebel et al, ), the development and assessment of gene knockdown/silencing methods (Adrianos, Lorenzen, & Oppert, ; Colinet et al, ; Dong & Friedrich, ; Fabrick, Kanost, & Baker, ; Khan, Reichelt, & Heckel, ; Perera, Little, & Pierce, ), and fertility and fecundity studies (Khanh, Bressac, & Chevrier, ; Pires, Abrão, Machado, Schofield, & Diotaiuti, ), and have been proposed as markers for field and dispersal monitoring (Shimizu & Kawasaki, ; Snodgrass, ), and as experimental models for aging (Campesan et al, ; Navrotskaya & Oxenkrug, ; Oxenkrug, ; Savvateeva et al, ).…”

“…Consequently, other techniques, such as the CRISPR/Cas9 system, could prove useful for demonstrating in vivo gene functionality. Although this technique has yet to be applied to Lygus bugs, previous studies have demonstrated the utility of targeting eye coloration genes as visible markers for developing CRISPR methods and/or screening phenotypic effects (Adrianos et al, ; Khan et al, ; F. Li & Scott, ; Perera et al, ; Xue et al, ). However, species‐specific functions of eye coloration genes that differ from those described in model systems have been reported (Khan et al, ).…”

CRISPR-Cas9-Based Genome Editing in the Silverleaf Whitefly (Bemisia tabaci)

“…Null mutants for this gene have white eyes in multiple taxa. [20][21][22] Cas9 proteins were complexed with five sgRNAs targeting exons 2, 3, and 5 of the gene ( Supplementary Table S1). We used saponin as an endosome escape reagent, as it was functional in Aedes aegypti and Anopheles gambiae ReMOT Control editing experiments.…”

Specific Gene Disruption in the Major Livestock PestsCochliomyia hominivoraxandLucilia cuprinaUsing CRISPR/Cas9

“…Due to its simplicity, efficiency and precision, CRISPR arises as a powerful genome editing technology, being successfully used to study gene function in vivo in a diversity of insects and other arthropods (see review by Sun et al (2017)). Typically, research projects target genes linked to evident phenotypes (Bassett et al 2013; Li and Scott 2016; Khan et al 2017; Li et al 2017b, 2017a; Heinze et al 2017; Meccariello et al 2017; Perera et al 2018; Choo et al 2018; Aumann et al 2018; Liu et al 2019; Sim et al 2019, to cite some examples), which allows a rapid and unambiguous screening for successful Cas9-mediated editing events. In addition to the sequenced genome of L. cuprina (Anstead et al 2015, 2016) and the current efforts to assemble and annotate the C. hominivorax genome (M.J. Scott, unpublished; A.C.M.…”