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

Authors: Perera, Omaththage; Little, Nathan; Pierce, Calvin

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/10/2018
Publication Date: 5/17/2018
Citation: Perera, O.P., Little, N., Pierce III, C.A. 2018. CRISPR/Cas9 mediated high efficiency knockout of the eye color gene vermillion in Helicoverpa zea (Boddie). PLoS One. 13(5):e0197567. https://doi.org/10.1371/journal.pone.0197567.
DOI: https://doi.org/10.1371/journal.pone.0197567

Interpretive Summary: Novel genome editing tools present opportunities to study various aspects of gene function (functional genomics) through specifically targeting genes or genomic regions of interest. In this study, clustered regularly interspersed palindromic repeats (CRISPR) and its associated nuclease (Cas) were used to evaluate efficiency of genome editing and optimize experimental conditions in a major crop pest, bollworm (Helicoverpa zea). Active ribonucleoprotein (RNP) complexes that target two specific nucleotide regions of the eye color gene vermilion were assembled from synthetic reagents and known concentrations were injected into freshly deposited eggs of bollworm. Change of eye color as well as mutations in the nucleotide sequences in the target gene region of injected bollworm adults were used to evaluate efficiency of genome editing with different concentrations of RNP complexes. We determined that embryo injections with RNP complex concentrations of 2 µM or higher produced significantly higher eye color mutants than those injected with 1 µM RNP complexes.

Technical 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 consists of ribonucleoprotein (RNP) complexes formed by combining guide RNA (gRNA) that target specific genomic 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 'M RNP complexes were compared using replicated experiments. Embryos injected with 2 or 4 'M RNP complexes displayed significantly higher mutation rates (>88%) in surviving adults compared to those injected with 1 'M. Hatch rates in embryos injected with RNP complexes and with injection buffer only (mock injections) was reduced by 19.8(±5.2)% compared to uninjected control embryos, but did not differ significantly between injected embryos. 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.