The CRISPR/Cas9 system is a fast, effective, and easy method for gene disruption, allowing generation of knockout animals by direct zygote injection. To date there is no report on the efficiency of this microinjection system in @entity291 zygotes and its effects on early development. The aim of this study was to compare 2 microinjection methods on developmental rates and efficiency to induce gene disruption. Microinjection effects on embryo development were evaluated by blastocyst (BL) formation rates at Day 8 of culture and by the proportion of lysed embryos (damaged during injection); while the efficiency of CRISPR/Cas9 RNA to create targeted mutations was studied by sequencing resulting blastocysts. Three groups were evaluated: (1) noninjected (control), (2) direct intracytoplasmic injection (direct-ICI), and (3) laser-assisted ICI (laser-ICI). Direct-ICI was performed with a beveled spiked glass needle (5 m ID; Origio, M  l  v, Denmark) to pass the @entity4091 ( @entity4091 ) and deliver CRISPR/Cas9 RNA as earlier described (Ross et al. 2008 BMC Dev. Biol. 8, 16). For laser-ICI, a Research Instruments (RI) Saturn 5 Active    laser system (Research Instruments Ltd., Falmouth, United Kingdom) was used to perforate the @entity4091 , and a blunt-end glass needle (5-6 m ID) used to deliver CRISPR/Cas9 RNA. In both cases, cytoplasm was aspirated into the pipette to disrupt the plasma membrane and the aspirated cytoplasm and CRISPR/Cas9 injected back into the embryo. Embryos were obtained by IVF of in vitro-matured oocytes aspirated from abattoir ovaries. At 18h post-IVF (hpf), zygotes were denuded from cumulus cells and cultured in groups of 25 in 50- L drops of KSOM (Evolve, Zenith Biotech, Guilford, CT, USA) with 4mgmL(-1) of BSA. Zygotes were injected at 20 to 22 hpf. Four biological replicates were assessed for BL rates (258 embryos total). A 2-tailed t-test was used to evaluate statistically significant differences (P <=0.05) between groups. Direct-ICI had a greater proportion of lysed embryos (29.5  10.6%) compared with laser-ICI (15.6  5%; P=0.056). BL development was significantly lower on direct-ICI (15.5  8%) compared with laser-ICI (31.4  5.9%; P=0.02) and control groups (32.8  6.6%; P=0.01). These results indicate that laser-ICI causes @entity16 and results in normal BL rates after microinjection (P=0.24). Because laser-ICI had normal BL rates and less embryo lysis, we used this method to evaluate efficiency of the CRISPR/Cas9 system to induce genomic mutations. Twelve BL were sequenced. Analysis of the CRISPR targeted region showed that 50% of the embryos had biallelic mutations, 33% monoallelic mutations, and 17% were wild type. These results show that laser-ICI method was very efficient for injecting CRISPR/Cas9 RNA into zygotes, resulting in normal developmental rates and a high amount of mutations in BL.

4 DEVELOPMENTAL OUTCOMES AND EFFICIENCY OF TWO CRISPR/Cas9 MICROINJECTION METHODS IN XXXX ZYGOTES.

@entity16 :: ('MESH:D009422', 'Disease') :: ['less damage'] @entity291 :: ('9913', 'Species') :: ['bovine'] @entity4091 :: ('MESH:D006562', 'Disease') :: ['zona pellucida', 'ZP']
@entity291 :: (9913,Species) :: ['BOVINE']
