Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos

Abstract

CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14-16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to early-stage injection (86.3%; 28.8%). However, gene editing was higher in blastocysts from late-(73.8%) vs. early-(63.8%) injected zygotes. Inhibition of the HR repair pathway increased gene editing efficiency by 15.6% in blastocysts from early-injected zygotes without compromising embryo development. Our finding shows that injection at the early cell cycle stage along with HR inhibition improves both zygote viability and gene editing rate in pig blastocysts.

Keywords: DNA repair; cell cycle; gene editing; pigs; zygotes.

Figure 1

Effect of early and late microinjection during the first cell cycle on embryo development. (A) Percentage of membrane lysis after early or late injections. (B) Cleavage and blastocyst rates after early or late injections in embryos from all technologies. (C) Cleavage and blastocyst rates after early or late injections for each embryo production technology. (D) Effect of early or late injections for targeting two gene sequences on cleavage and blastocyst rates of parthenogenetic-activated embryos. Early injections correspond to 0–6 h post activation/fertilization (before/early S-phase). Late injections correspond to 14–16 h post activation/fertilization (late/after S-phase). PA, parthenogenetic activation; SCNT, somatic cell nuclear transfer; IVF, in vitro fertilization; and ICSI, intracytoplasmic sperm injection. Asterisk represents statistical differences (p < 0.05). At least six replicates were performed by each embryo production system, containing 30–35 oocytes.

Figure 2

Effect of early and late microinjection during the first cell cycle on gene editing rates. (A) Percentage of gene-edited blastocysts from each embryo production technology. (B) Percentage of gene-edited blastocysts after early or late microinjections in embryos from all technologies. (C) Percentage of gene-edited blastocysts after early or late injections for each embryo production technology. Early injections correspond to 0–6 h post activation/fertilization (before/early S-phase). Late injections correspond to 14–16 h post activation/fertilization (late/after S-phase). PA, parthenogenetic activation; SCNT, somatic cell nuclear transfer; IVF, in vitro fertilization; and ICSI, intracytoplasmic sperm injection. * represents statistical differences (p < 0.05) and # represents a tendency (p = 0.07). In total, 301 embryos were evaluated (before/early S-phase [n = 143]: PA [n = 71], SCNT [n = 25], IVF [n = 21], ICSI [n = 26]; after/late S-phase [n = 158]: PA [n = 94], SCNT [n = 24], IVF [n = 25], ICSI [n = 15]).

Figure 3

Interaction between time of microinjection and homologous recombination inhibition on embryo development. Cleavage and blastocyst rates after early and late microinjections and treated (ATMi) or not treated (CT) with an inhibitor of homologous recombination in embryos produced by different technologies. Early injections correspond to 0–6 h post activation/fertilization (before/early S-phase). Late injections correspond to 14–16 h post activation/fertilization (late/after S-phase). PA, parthenogenetic activation; SCNT, somatic cell nuclear transfer; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; ATMi, treated with KU-55933 an ATM inhibitor; and homologous recombination pathway). Capital letters indicate statistical differences on cleavage rates, and lowercase letters indicate statistical differences on blastocyst rates (p < 0.05). At least six replicates were performed by each embryo production system, containing 30–35 oocytes.

Figure 4

Interaction between time of injection and inhibition of homologous recombination on gene-editing rates. (A) Percentage of gene-edited blastocysts after early or late microinjection followed by treatment (ATMi) or not (CT) with an inhibitor of homologous recombination in embryos from all technologies. (B) Percentage of gene-edited blastocysts after early or late microinjection followed by treatment (ATMi) or not (CT). Early injections correspond to 0–6 h post activation/fertilization (before/early S-phase). Late injections correspond to 14–16 h post activation/fertilization (late/after S-phase). PA, parthenogenetic activation; SCNT, somatic cell nuclear transfer; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; and ATMi, treated with KU-55933 (an ATM inhibitor; homologous recombination pathway). * represents statistical differences (p < 0.05) and # represents a tendency (p = 0.07). In total, 571 embryos were evaluated (Early-CT [n = 143]: PA [n = 71], SCNT [n = 25], IVF [n = 21], ICSI [n = 26]; Early-ATMi [n = 139]: PA [n = 77], SCNT [n = 18], IVF [n = 25], ICSI [n = 19]; Late-CT [n = 158]: PA [n = 94], SCNT [n = 24], IVF [n = 25], ICSI [n = 15]; Late-ATMi [n = 131]: PA [n = 78], SCNT [n = 23], IVF [n = 20], ICSI [n = 10]).

Figure 5

Effect of time of microinjection and homologous recombination inhibition on the rate of double gene editing in embryos produced by parthenogenetic activation. (A) Percentage of double gene editing after early or late microinjections. (B) Percentage of double gene editing after early or late stages followed by treatment (ATMi) or not (CT) with an ATM inhibitor. ATMi, treated with KU-55933 (an ATM inhibitor; homologous recombination pathway). In total, 72 embryos were analyzed (Early-CT [n = 18], Early-ATMi [n = 18], Late-CT [n = 18], Late-ATMi [n = 18]).