GENERATION OF MULTIPLE KNOCKOUTS FOR CYSTEIN-RICH SECRETORY PROTEINS USING CRISPR/ CAS9 TECHNOLOGY

CURCI L; DA ROS VG; ROJO D; CUASNICU PS; BRUKMAN N; RUBINSTEIN M

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Cysteine-Rich Secretory Protein (CRISP) 1, 2, 3 and 4 aremainly expressed in the reproductive tract and have key roles inmammalian fertilization. In spite of this, mutant mice lacking eitherCRISP1, 2 or 4 are fertile. To investigate the functional relevanceof CRISP3, we developed Crisp3 knockout mice using the novelCRISPR/Cas9 technique. With the idea of also generating doubleCrisp1-/-/Crisp3-/- mice, sgRNAs targeting exon 2 of Crisp1 andCrisp3 were selected by bioinformatic analysis. These sgRNAs weremicroinjected together with Cas9 mRNA into C57BL/6 mouse zygotesand then transferred to pseudopregnant females. Mice carryingthe targeted mutations were identified by PCR of genomic DNA andSDS-PAGE. Germline transmission of the mutations was confirmedby DNA sequencing and mice carrying heterozygous null-allele mutationsin Crisp3 or in Crisp1 and Crisp3 were selected as breedersto obtain homozygous mutants. Expression analysis of CRISPproteins in mutant mice performed by Western blot revealed thatboth Crisp3-/- and Crisp1-/-/Crisp3-/- animals lack CRISP1 whereasCRISP2 and CRISP4 were not affected in either colony. Fertility ratesevaluated by natural mating were significantly reduced in maleand female mice from both colonies, suggesting that the normal fertilityobserved in single knockouts involves compensatory mechanismsbetween homologous CRISP proteins. To generate mice simultaneouslylacking the four CRISP family members, we microinjectedsgRNAs targeting Crisp1 and Crisp3 into Crisp2-/-/Crisp4+/- zygotes.Recent results confirmed the successful generation of mice with nullalleles of the four Crisp genes. We believe these studies will contributeto a better understanding of the relevance of this importantfamily for animal fertility.