Supplementary Data

Comparison of in vitro embryo and piglet editing frequency

For RELA TALEN mRNA injected zygotes we transferred 393 embryos into 11 recipient sows, resulting in 6 pregnancies and 5 farrowings with litter sizes ranging from 3 to 17. One female aborted within the last two weeks of pregnancy. Of the 46 piglets born, 5 were stillborn while of the live born 13 were savaged, sat on by the sow or culled on veterinary advice. Post mortem investigation failed to detect any common pathology associated with the dead piglets.

In comparison to previous studies describing the combination of genome editing tools and SCNT, zygote injection represents an efficient technology. In our work described here 32 donor/recipient animals were used to produce 9 edited piglets. In contrast the generation of genome edited pigs by SCNT used, for example, approximately 75 donor/recipients to produce 2 edited animals12 while approximately 84 donor/recipient animals were used to produce 11 edited piglets in a report of biallelic genome editing10; assuming 20 oocytes per donor animal.

Supplementary Fig 1. Example TALEN edited pig (#26) carrying single base deletion with sequence read of single base pair edited allele and wild-type RELA allele.

TALEN binding site

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

Zygote 1

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT GGTGTATCCATGCCCCCCCACACAGCTGAGCCC~~~~TGATGGAGTACCCTGAGGCTATAACTC4

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTAGTGGAGTACCCTGAGGCTATAACTC2+2

Zygote 2

GGTGTATCCATGCCCCCCCACACAGCTGAG~CCATGCTGATGGAGTACCCTGAGGCTATAACTC1

Zygote 3

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~~~~~ATGGAGTACCCTGAGGCTATAACTC5

Zygote 4

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATTGCTGATGGAGTACCCTGAGGCTATAACT+1

Zygote 5

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT GGTGTATCCATGCCCCCCCACACAGCTGAGCCC~TGCTGATGGAGTACCCTGAGGCTATAACTC1

Zygote 6

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT GGTGTATCCATGCCCCCCCACACAGCTGAGCCC~~~~TGATGGAGTACCCTGAGGCTATAACTC4

Zygote 7

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCTGAGGCTATAACTC1

Zygote 8

GGTGTATCCATGCCCCCCCACACAGCTGAGCC35TGCTGATGGAGTACCCTGAGGCTATAACTC2+35

ACAAGTCGAGGGTCTCAGAGTCACTGAGTACTGAG

Zygote 9

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTACCCATGCCCCCCCACACAGCTGAGCCCA~~~~~ATGGAGTACCCTGAGGCTATAACTC5

Zygote 10

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCT~~~~GATGGAGTACCCTGAGGCTATAACTC 4

Zygote 11

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT GGTGTATCCATGCCCCCCCACACAGCTGAGCC~ATGCTGATGGAGTACCCTGAGGCTATAACTC1

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGG~~~~CCCTGAGGCTATAACTC4

Zygote 12

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT GGTGTATCCATGCCCCCCCACACAGCTGAGCC~ATGCTGATGGAGTACCCTGAGGCTATAACTC 1

Zygote 13

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATG~TGATGGAGTACCCTGAGGCTATAACTC1

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCAT~~TGATGGAGTACCCTGAGGCTATAACTC 2

Zygote 14

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCTGAGCC~ATGCTGATGGAGTACCCTGAGGCTATAACTC1

Zygote 15

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCTGAGCCC~~~~TGATGGAGTACCCTGAGGCTATAACTC4

Zygote 16

GGTGTATCCATGCCCCCCCACACAGCTGAGC~~ACACA~~~GGAGTACCCTGAGGCTATAACTC10+5

Zygote 17

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCT~~~~~~~~~~~GATGGAGTACCCTGAGGCTATAACTC 11

Zygote 18

GGTGTATCCATGCCCCCCCACACAGCT~~~~~~~~~~~~~~~GAGTACCCTGAGGCTATAACTC 15

Zygote 19

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~~~TGATGGAGTACCCTGAGGCTATAACTC 3

Zygote 20

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCTGA~~~~~~GCTGATGGAGTACCCTGAGGCTATAACTC 6

Zygote 21

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCACAGCTGGTACTCAGATGGAGTACCCTGAGGCTATAACT +9

GGTGTATCCATGCCCCCCCACACAGCT~~~~~~~~~~~GATGGAGTACCCTGAGGCTATAACTC 11

SupplementaryFig 2. Sequence of indels identified in zygotes. Zygotes 1-16 were injected with 20ng/ul TALEN mRNA and screened for GFP. Zygotes 17 & 18were injected with 20ng/ul TALEN mRNA and were screened for development. Zygotes 19 – 21 were injected with 2ng/ul TALEN mRNA.

TALEN binding site

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

Piglet 8770-I

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCTGAGGCTATAACTC 1

Piglet 8770-J

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATTGCTGATGGAGTACCCTGAGGCTATAACT +1

Piglet 8770-26

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCTGAGGCTATAACTC 1

Piglet 8130-so

GGTGTATCCATGCCCCCCCACACAGCTGA~~~~~~GCTGATGGAGTACCCTGAGGCTATAACTC 6

Piglet 8130-16

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTC WT

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCTCCATCAGCTGATGGAGTACCCTGAGGCTAT +5

GGTGTATCCATGCCCCCCCACACAGCTGAG~~~~~~~~~~~~~~~~~CCCTGAGGCTATAACTC 17

Inserted sequence is duplication/inversion of orange highlighted

Piglet 8784-30

GGTGTATCCATGCCCCCCCACACAGCTGAGC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 111

GGTGTATCCATGCCCCCCCACACAGCTGAGCC~~~~CTGATGGAGTACCCTGAGGCTATAACTC 4

Piglet 8784-33

GGTGTATCCATGCCCCCCCACACAGCTGAGC~~~~~CTGATGGAGTACCCTGAGGCTATAACTC 5

GGTGTATCCATGCCCCCCCACACAGCTGAG~~~~~~CTGATGGAGTACCCTGAGGCTATAACTC 6

GGTGTATCCATGCCCCCCCACACAGCTGAGCC~~~~CTGATGGAGTACCCTGAGGCTATAACTC 4

Piglet 8784-34

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCTGAGGCTATAACTC 1

GGTGTATCCATGCCCCCCCACACAGCTGAGCCCTGAGTACCCTGAGGAGTACCCTGAGGCTATA 8+12

Inserted sequence is duplication of orange highlighted

ZFN binding site

TGCTGATGGAGTACCCTGAGGCTATAACTCGCTTGGTGACAGGGTCCCAGAGACCCCCTGACCWT

Piglet 8142-C

TGCTGATGGAGTACCCTGAGGCTATAACTC~~~~~~~~~TCTGGGA~~~~~~~~~CCCTGACC25+7

Inserted sequence is inversion of orange highlighted in consensus

SupplementaryFig 3. Sequence of indels identified in piglets.