Using gene editors in sheep embryos allows efficient single gene changes but shows challenges when editing multiple genes at once

High editing efficiencies of 95.0% for BMPR1B and 65.0% for SOCS2 were achieved using a zygote electroporation platform.

• Bystander edits were prevalent despite high editing efficiencies in single-target validation.
• Dual-target editing introduced guide RNA crosstalk between different base editors in embryos.
• Three live lambs were obtained from dual-editing blastocysts, all carrying the intended BMPR1B mutation.
• Bystander edits consistently accompanied the desired mutations in the lambs.
• One lamb showed additional gRNA crosstalk and an unintended deletion at the SOCS2 locus, leading to a predicted null allele.
• Multiplex base editing systems face significant challenges for routine application in sheep breeding due to unpredictable genomic outcomes.

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