Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes

TY - JOUR

T1 - Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes

AU - Meccariello, Angela

AU - Monti, Simona Maria

AU - Romanelli, Alessandra

AU - Colonna, Rita

AU - Primo, Pasquale

AU - Inghilterra, Maria Grazia

AU - Del Corsano, Giuseppe

AU - Ramaglia, Antonio

AU - Iazzetti, Giovanni

AU - Chiarore, Antonia

AU - Patti, Francesco

AU - Heinze, Svenia D.

AU - Salvemini, Marco

AU - Lindsay, Helen

AU - Chiavacci, Elena

AU - Burger, Alexa

AU - Robinson, Mark D.

AU - Mosimann, Christian

AU - Bopp, Daniel

AU - Saccone, Giuseppe

PY - 2017/12/1

Y1 - 2017/12/1

N2 - The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

AB - The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

UR - https://www.scopus.com/pages/publications/85028524093

U2 - 10.1038/s41598-017-10347-5

DO - 10.1038/s41598-017-10347-5

M3 - Article

C2 - 28855635

AN - SCOPUS:85028524093

SN - 2045-2322

VL - 7

JO - Scientific Reports

JF - Scientific Reports

IS - 1

M1 - 10061

ER -