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[PDF] What if gene editing became routine practice? - European Parliament
genetic modification of human embryos using CRISPR-Cas91 and related techniques, for research Gene editing is a rapidly developing area of biotechnology
[PDF] History of genome editing: From meganucleases to CRISPR
CRISPR-mediated genome editing has undoubtedly revolutionized genetic engineering of animals With the ability for virtually unlimited modification of
[PDF] Advanced Gene Editing: CRISPR-Cas9
7 déc 2018 · Prior use of CRISPR-Cas9 gene editing in human embryos was generally limited to non- viable embryos, in part, to address ethical concerns
[PDF] GENE EDITING MYTHS AND REALITY - Greens/EFA
engineered with gene editing excluded from them These Also, CRISPR gene editing of rice plants was editing is necessary and critical before a new
Cas9 technology in the human germline and pluripotent stem cells
29 fév 2016 · until recently, DNA editing has been virtually unviable when targeting editing technologies and human germline genetic modification, www
![[PDF] History of genome editing: From meganucleases to CRISPR](https://pdfprof.com/EN_PDFV2/Docs/PDF_3/117076_3Manuscript_KUPS_publish.pdf.jpg "[PDF] History of genome editing: From meganucleases to CRISPR")
117076_3Manuscript_KUPS_publish.pdf History of genome editingrom meganucleases to CRISPR Simon E. Tröder Sup and Branko Zevnik
Supl S in vivoResearch Facility
, Faculty of Medicine and University Hospital Cologne ,University
of Cologne,Cologne, Germany
p Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany lCorresponding author
E - mail: branko.zevnik@uk - koeln.de 19:plm02lt
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[D06D0WM 7/84?/0W 8737W H01,6 H0 Supplementary material for
History of genome editing - from meganucleases to CRISPR Simon E. Tröder and Branko Zevnik
Table S1. Supplementary references for in vivo genome editing Method Species Reference
ZFN mouse 1, 2 rat 3 rabbit 4 pig 5, 6 cattle 7, 8 TALEN
worm 9 zebrafish 10, 11
frog 12 rat 13 mouse 14-16 rabbit 17 pig 18, 19
goat 20 sheep 21
cattle 21
non-human primate 22
CRISPR/Cas9
fly 23
worm 24
zebrafish 25
frog 26
rat 27
rabbit 28
pig 29
cattle 30
goat 31
sheep 32
non-human primate 33
CRISPR base editing
zebrafish 34
frog 35
mouse 36
rat 37, 38
rabbit 39
pig 40
goat 41
sheep 42
non-human primate 43
Supplementary references
1. Meyer M, de Angelis MH, Wurst W, et al. Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A 2010; 107: 15022-15026. 2010/08/06. DOI: 10.1073/pnas.1009424107. 2. Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using
zinc-finger nucleases. Genetics 2010; 186: 451-459. 2010/07/16. DOI: 10.1534/genetics.110.117002.
3. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of
zinc-finger nucleases. Science 2009; 325: 433. 2009/07/25. DOI: 10.1126/science.1172447.
4. Flisikowska T, Thorey IS, Offner S, et al. Efficient immunoglobulin gene disruption
and targeted replacement in rabbit using zinc finger nucleases. PloS one 2011; 6: e21045. 2011/06/23. DOI: 10.1371/journal.pone.0021045. 5. Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout
in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12017. 2011/07/07. DOI: 10.1073/pnas.1106422108.
6. Whyte JJ, Zhao J, Wells KD, et al. Gene targeting with zinc finger nucleases to
produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78: 2. 2011/01/27. DOI: 10.1002/mrd.21271.
7. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows. Nat Commun 2013; 4: 2565. 2013/10/15. DOI: 10.1038/ncomms3565.
8. Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG)
gene via zinc-finger nucleases in cattle. Cell research 2011; 21: 1638-1640. 2011/09/14. DOI: 10.1038/cr.2011.153.
9. Wood AJ, Lo TW, Zeitler B, et al. Targeted genome editing across species using ZFNs
and TALENs. Science 2011; 333: 307. 2011/06/28. DOI: 10.1126/science.1207773. 10. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using
customized TALENs. Nat Biotechnol 2011; 29: 699-700. 2011/08/09. DOI: 10.1038/nbt.1939.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish
cells using engineered TALENs. Nat Biotechnol 2011; 29: 697-698. 2011/08/09. DOI: 10.1038/nbt.1934.
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using
engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012; 109: 17484-17489. 2012/10/10. DOI: 10.1073/pnas.1215421109. 13. Tesson L, Usal C, Menoret S, et al. Knockout rats generated by embryo microinjection
of TALENs. Nat Biotechnol 2011; 29: 695-696. 2011/08/09. DOI: 10.1038/nbt.1940. 14. Panda SK, Wefers B, Ortiz O, et al. Highly efficient targeted mutagenesis in mice
using TALENs. Genetics 2013; 195: 703-713. 2013/08/28. DOI: 10.1534/genetics.113.156570.
15. Sung YH, Baek IJ, Kim DH, et al. Knockout mice created by TALEN-mediated gene
targeting. Nat Biotechnol 2013; 31: 23-24. 2013/01/11. DOI: 10.1038/nbt.2477. 16. Wefers B, Panda SK, Ortiz O, et al. Generation of targeted mouse mutants by embryo
microinjection of TALEN mRNA. Nature protocols 2013; 8: 2355-2379. 2013/11/02. DOI: 10.1038/nprot.2013.142.
17. Song J, Zhong J, Guo X, et al. Generation of RAG 1- and 2-deficient rabbits by embryo
microinjection of TALENs. Cell research 2013; 23: 1059-1062. 2013/07/10. DOI: 10.1038/cr.2013.85.
18. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in
livestock. Proc Natl Acad Sci U S A 2012; 109: 17382-17387. 2012/10/03. DOI: 10.1073/pnas.1211446109.
19. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited
zygotes. Sci Rep 2013; 3: 2847. 2013/10/11. DOI: 10.1038/srep02847. 20. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous
recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 2015; 5: 10482. 2015/05/23. DOI: 10.1038/srep10482. 21. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle.
Transgenic research 2015; 24: 147-153. 2014/09/11. DOI: 10.1007/s11248-014-9832- x. 22. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and
cynomolgus monkeys. Cell Stem Cell 2014; 14: 323-328. 2014/02/18. DOI: 10.1016/j.stem.2014.01.018.
23. Bassett AR, Tibbit C, Ponting CP, et al. Highly efficient targeted mutagenesis of
Drosophila with the CRISPR/Cas9 system. Cell Rep 2013; 4: 220-228. 2013/07/06. DOI: 10.1016/j.celrep.2013.06.020.
24. Friedland AE, Tzur YB, Esvelt KM, et al. Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nature methods 2013; 10: 741-743. 2013/07/03. DOI: 10.1038/nmeth.2532.
25. Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-
Cas system. Nat Biotechnol 2013; 31: 227-229. 2013/01/31. DOI: 10.1038/nbt.2501. 26. Nakayama T, Fish MB, Fisher M, et al. Simple and efficient CRISPR/Cas9-mediated
targeted mutagenesis in Xenopus tropicalis. Genesis 2013; 51: 835-843. 2013/10/15. DOI: 10.1002/dvg.22720.
27. Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of
multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 2013; 31: 684-686. 2013/08/10. DOI: 10.1038/nbt.2652.
28. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9
nucleases. J Mol Cell Biol 2014; 6: 97-99. 2014/01/10. DOI: 10.1093/jmcb/mjt047. 29. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection
of CRISPR/Cas system. Cell research 2014; 24: 372-375. 2014/02/01. DOI: 10.1038/cr.2014.11.
30. Bevacqua RJ, Fernandez-Martin R, Savy V, et al. Efficient edition of the bovine PRNP
prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology 2016; 86: 1886-1896 e1881. 2016/08/28. DOI: 10.1016/j.theriogenology.2016.06.010.
31. Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and
FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 2015; 5: 13878. 2015/09/12. DOI: 10.1038/srep13878.
32. Crispo M, Mulet AP, Tesson L, et al. Efficient Generation of Myostatin Knock-Out
Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PloS one 2015; 10: e0136690. 2015/08/26. DOI: 10.1371/journal.pone.0136690.
33. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via
Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156: 836-843. 2014/02/04. DOI: 10.1016/j.cell.2014.01.027.
34. Zhang Y, Qin W, Lu X, et al. Programmable base editing of zebrafish genome using a
modified CRISPR-Cas9 system. Nat Commun 2017; 8: 118. 2017/07/26. DOI: 10.1038/s41467-017-00175-6.
35. Park DS, Yoon M, Kweon J, et al. Targeted Base Editing via RNA-Guided Cytidine
Deaminases in Xenopus laevis Embryos. Mol Cells 2017; 40: 823-827. 2017/11/28. DOI: 10.14348/molcells.2017.0262.
36. Kim K, Ryu SM, Kim ST, et al. Highly efficient RNA-guided base editing in mouse
embryos. Nat Biotechnol 2017; 35: 435-437. 2017/03/01. DOI: 10.1038/nbt.3816. 37. Ma Y, Yu L, Zhang X, et al. Highly efficient and precise base editing by engineered
dCas9-guide tRNA adenosine deaminase in rats. Cell Discov 2018; 4: 39. 2018/07/25. DOI: 10.1038/s41421-018-0047-9.
38. Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors
in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 2018; 9: 814-819. 2018/08/02. DOI: 10.1007/s13238-018-0568-x. 39. Liu Z, Chen M, Chen S, et al. Highly efficient RNA-guided base editing in rabbit. Nat
Commun 2018; 9: 2717. 2018/07/15. DOI: 10.1038/s41467-018-05232-2. 40. Xie J, Ge W, Li N, et al. Efficient base editing for multiple genes and loci in pigs using
base editors. Nat Commun 2019; 10: 2852. 2019/06/30. DOI: 10.1038/s41467-019- 10421-8.
41. Li G, Zhou S, Li C, et al. Base pair editing in goat: nonsense codon introgression into
FGF5 results in longer hair. The FEBS journal 2019; 286: 4675-4692. 2019/07/06. DOI: 10.1111/febs.14983.
42. Zhou S, Cai B, He C, et al. Programmable Base Editing of the Sheep Genome Revealed
No Genome-Wide Off-Target Mutations. Front Genet 2019; 10: 215. 2019/04/02. DOI: 10.3389/fgene.2019.00215.
43. Zhang W, Aida T, Del Rosario RCH, et al. Multiplex precise base editing in cynomolgus
monkeys. Nat Commun 2020; 11: 2325. 2020/05/13. DOI: 10.1038/s41467-020- 16173-0.
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[D06D0WM 7/84?/0W 8737W H01,6 H0 Supplementary material for
History of genome editing - from meganucleases to CRISPR Simon E. Tröder and Branko Zevnik
Table S1. Supplementary references for in vivo genome editing Method Species Reference
ZFN mouse 1, 2 rat 3 rabbit 4 pig 5, 6 cattle 7, 8 TALEN
worm 9 zebrafish 10, 11
frog 12 rat 13 mouse 14-16 rabbit 17 pig 18, 19
goat 20 sheep 21
cattle 21
non-human primate 22
CRISPR/Cas9
fly 23
worm 24
zebrafish 25
frog 26
rat 27
rabbit 28
pig 29
cattle 30
goat 31
sheep 32
non-human primate 33
CRISPR base editing
zebrafish 34
frog 35
mouse 36
rat 37, 38
rabbit 39
pig 40
goat 41
sheep 42
non-human primate 43
Supplementary references
1. Meyer M, de Angelis MH, Wurst W, et al. Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A 2010; 107: 15022-15026. 2010/08/06. DOI: 10.1073/pnas.1009424107. 2. Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using
zinc-finger nucleases. Genetics 2010; 186: 451-459. 2010/07/16. DOI: 10.1534/genetics.110.117002.
3. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of
zinc-finger nucleases. Science 2009; 325: 433. 2009/07/25. DOI: 10.1126/science.1172447.
4. Flisikowska T, Thorey IS, Offner S, et al. Efficient immunoglobulin gene disruption
and targeted replacement in rabbit using zinc finger nucleases. PloS one 2011; 6: e21045. 2011/06/23. DOI: 10.1371/journal.pone.0021045. 5. Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout
in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12017. 2011/07/07. DOI: 10.1073/pnas.1106422108.
6. Whyte JJ, Zhao J, Wells KD, et al. Gene targeting with zinc finger nucleases to
produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78: 2. 2011/01/27. DOI: 10.1002/mrd.21271.
7. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows. Nat Commun 2013; 4: 2565. 2013/10/15. DOI: 10.1038/ncomms3565.
8. Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG)
gene via zinc-finger nucleases in cattle. Cell research 2011; 21: 1638-1640. 2011/09/14. DOI: 10.1038/cr.2011.153.
9. Wood AJ, Lo TW, Zeitler B, et al. Targeted genome editing across species using ZFNs
and TALENs. Science 2011; 333: 307. 2011/06/28. DOI: 10.1126/science.1207773. 10. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using
customized TALENs. Nat Biotechnol 2011; 29: 699-700. 2011/08/09. DOI: 10.1038/nbt.1939.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish
cells using engineered TALENs. Nat Biotechnol 2011; 29: 697-698. 2011/08/09. DOI: 10.1038/nbt.1934.
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using
engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012; 109: 17484-17489. 2012/10/10. DOI: 10.1073/pnas.1215421109. 13. Tesson L, Usal C, Menoret S, et al. Knockout rats generated by embryo microinjection
of TALENs. Nat Biotechnol 2011; 29: 695-696. 2011/08/09. DOI: 10.1038/nbt.1940. 14. Panda SK, Wefers B, Ortiz O, et al. Highly efficient targeted mutagenesis in mice
using TALENs. Genetics 2013; 195: 703-713. 2013/08/28. DOI: 10.1534/genetics.113.156570.
15. Sung YH, Baek IJ, Kim DH, et al. Knockout mice created by TALEN-mediated gene
targeting. Nat Biotechnol 2013; 31: 23-24. 2013/01/11. DOI: 10.1038/nbt.2477. 16. Wefers B, Panda SK, Ortiz O, et al. Generation of targeted mouse mutants by embryo
microinjection of TALEN mRNA. Nature protocols 2013; 8: 2355-2379. 2013/11/02. DOI: 10.1038/nprot.2013.142.
17. Song J, Zhong J, Guo X, et al. Generation of RAG 1- and 2-deficient rabbits by embryo
microinjection of TALENs. Cell research 2013; 23: 1059-1062. 2013/07/10. DOI: 10.1038/cr.2013.85.
18. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in
livestock. Proc Natl Acad Sci U S A 2012; 109: 17382-17387. 2012/10/03. DOI: 10.1073/pnas.1211446109.
19. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited
zygotes. Sci Rep 2013; 3: 2847. 2013/10/11. DOI: 10.1038/srep02847. 20. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous
recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 2015; 5: 10482. 2015/05/23. DOI: 10.1038/srep10482. 21. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle.
Transgenic research 2015; 24: 147-153. 2014/09/11. DOI: 10.1007/s11248-014-9832- x. 22. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and
cynomolgus monkeys. Cell Stem Cell 2014; 14: 323-328. 2014/02/18. DOI: 10.1016/j.stem.2014.01.018.
23. Bassett AR, Tibbit C, Ponting CP, et al. Highly efficient targeted mutagenesis of
Drosophila with the CRISPR/Cas9 system. Cell Rep 2013; 4: 220-228. 2013/07/06. DOI: 10.1016/j.celrep.2013.06.020.
24. Friedland AE, Tzur YB, Esvelt KM, et al. Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nature methods 2013; 10: 741-743. 2013/07/03. DOI: 10.1038/nmeth.2532.
25. Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-
Cas system. Nat Biotechnol 2013; 31: 227-229. 2013/01/31. DOI: 10.1038/nbt.2501. 26. Nakayama T, Fish MB, Fisher M, et al. Simple and efficient CRISPR/Cas9-mediated
targeted mutagenesis in Xenopus tropicalis. Genesis 2013; 51: 835-843. 2013/10/15. DOI: 10.1002/dvg.22720.
27. Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of
multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 2013; 31: 684-686. 2013/08/10. DOI: 10.1038/nbt.2652.
28. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9
nucleases. J Mol Cell Biol 2014; 6: 97-99. 2014/01/10. DOI: 10.1093/jmcb/mjt047. 29. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection
of CRISPR/Cas system. Cell research 2014; 24: 372-375. 2014/02/01. DOI: 10.1038/cr.2014.11.
30. Bevacqua RJ, Fernandez-Martin R, Savy V, et al. Efficient edition of the bovine PRNP
prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology 2016; 86: 1886-1896 e1881. 2016/08/28. DOI: 10.1016/j.theriogenology.2016.06.010.
31. Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and
FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 2015; 5: 13878. 2015/09/12. DOI: 10.1038/srep13878.
32. Crispo M, Mulet AP, Tesson L, et al. Efficient Generation of Myostatin Knock-Out
Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PloS one 2015; 10: e0136690. 2015/08/26. DOI: 10.1371/journal.pone.0136690.
33. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via
Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156: 836-843. 2014/02/04. DOI: 10.1016/j.cell.2014.01.027.
34. Zhang Y, Qin W, Lu X, et al. Programmable base editing of zebrafish genome using a
modified CRISPR-Cas9 system. Nat Commun 2017; 8: 118. 2017/07/26. DOI: 10.1038/s41467-017-00175-6.
35. Park DS, Yoon M, Kweon J, et al. Targeted Base Editing via RNA-Guided Cytidine
Deaminases in Xenopus laevis Embryos. Mol Cells 2017; 40: 823-827. 2017/11/28. DOI: 10.14348/molcells.2017.0262.
36. Kim K, Ryu SM, Kim ST, et al. Highly efficient RNA-guided base editing in mouse
embryos. Nat Biotechnol 2017; 35: 435-437. 2017/03/01. DOI: 10.1038/nbt.3816. 37. Ma Y, Yu L, Zhang X, et al. Highly efficient and precise base editing by engineered
dCas9-guide tRNA adenosine deaminase in rats. Cell Discov 2018; 4: 39. 2018/07/25. DOI: 10.1038/s41421-018-0047-9.
38. Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors
in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 2018; 9: 814-819. 2018/08/02. DOI: 10.1007/s13238-018-0568-x. 39. Liu Z, Chen M, Chen S, et al. Highly efficient RNA-guided base editing in rabbit. Nat
Commun 2018; 9: 2717. 2018/07/15. DOI: 10.1038/s41467-018-05232-2. 40. Xie J, Ge W, Li N, et al. Efficient base editing for multiple genes and loci in pigs using
base editors. Nat Commun 2019; 10: 2852. 2019/06/30. DOI: 10.1038/s41467-019- 10421-8.
41. Li G, Zhou S, Li C, et al. Base pair editing in goat: nonsense codon introgression into
FGF5 results in longer hair. The FEBS journal 2019; 286: 4675-4692. 2019/07/06. DOI: 10.1111/febs.14983.
42. Zhou S, Cai B, He C, et al. Programmable Base Editing of the Sheep Genome Revealed
No Genome-Wide Off-Target Mutations. Front Genet 2019; 10: 215. 2019/04/02. DOI: 10.3389/fgene.2019.00215.
43. Zhang W, Aida T, Del Rosario RCH, et al. Multiplex precise base editing in cynomolgus
monkeys. Nat Commun 2020; 11: 2325. 2020/05/13. DOI: 10.1038/s41467-020- 16173-0.
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[D06D0WM 7/84?/0W 8737W H01,6 H0 Supplementary material for
History of genome editing - from meganucleases to CRISPR Simon E. Tröder and Branko Zevnik
Table S1. Supplementary references for in vivo genome editing Method Species Reference
ZFN mouse 1, 2 rat 3 rabbit 4 pig 5, 6 cattle 7, 8 TALEN
worm 9 zebrafish 10, 11
frog 12 rat 13 mouse 14-16 rabbit 17 pig 18, 19
goat 20 sheep 21
cattle 21
non-human primate 22
CRISPR/Cas9
fly 23
worm 24
zebrafish 25
frog 26
rat 27
rabbit 28
pig 29
cattle 30
goat 31
sheep 32
non-human primate 33
CRISPR base editing
zebrafish 34
frog 35
mouse 36
rat 37, 38
rabbit 39
pig 40
goat 41
sheep 42
non-human primate 43
Supplementary references
1. Meyer M, de Angelis MH, Wurst W, et al. Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A 2010; 107: 15022-15026. 2010/08/06. DOI: 10.1073/pnas.1009424107. 2. Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using
zinc-finger nucleases. Genetics 2010; 186: 451-459. 2010/07/16. DOI: 10.1534/genetics.110.117002.
3. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of
zinc-finger nucleases. Science 2009; 325: 433. 2009/07/25. DOI: 10.1126/science.1172447.
4. Flisikowska T, Thorey IS, Offner S, et al. Efficient immunoglobulin gene disruption
and targeted replacement in rabbit using zinc finger nucleases. PloS one 2011; 6: e21045. 2011/06/23. DOI: 10.1371/journal.pone.0021045. 5. Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout
in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12017. 2011/07/07. DOI: 10.1073/pnas.1106422108.
6. Whyte JJ, Zhao J, Wells KD, et al. Gene targeting with zinc finger nucleases to
produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78: 2. 2011/01/27. DOI: 10.1002/mrd.21271.
7. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows. Nat Commun 2013; 4: 2565. 2013/10/15. DOI: 10.1038/ncomms3565.
8. Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG)
gene via zinc-finger nucleases in cattle. Cell research 2011; 21: 1638-1640. 2011/09/14. DOI: 10.1038/cr.2011.153.
9. Wood AJ, Lo TW, Zeitler B, et al. Targeted genome editing across species using ZFNs
and TALENs. Science 2011; 333: 307. 2011/06/28. DOI: 10.1126/science.1207773. 10. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using
customized TALENs. Nat Biotechnol 2011; 29: 699-700. 2011/08/09. DOI: 10.1038/nbt.1939.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish
cells using engineered TALENs. Nat Biotechnol 2011; 29: 697-698. 2011/08/09. DOI: 10.1038/nbt.1934.
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using
engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012; 109: 17484-17489. 2012/10/10. DOI: 10.1073/pnas.1215421109. 13. Tesson L, Usal C, Menoret S, et al. Knockout rats generated by embryo microinjection
of TALENs. Nat Biotechnol 2011; 29: 695-696. 2011/08/09. DOI: 10.1038/nbt.1940. 14. Panda SK, Wefers B, Ortiz O, et al. Highly efficient targeted mutagenesis in mice
using TALENs. Genetics 2013; 195: 703-713. 2013/08/28. DOI: 10.1534/genetics.113.156570.
15. Sung YH, Baek IJ, Kim DH, et al. Knockout mice created by TALEN-mediated gene
targeting. Nat Biotechnol 2013; 31: 23-24. 2013/01/11. DOI: 10.1038/nbt.2477. 16. Wefers B, Panda SK, Ortiz O, et al. Generation of targeted mouse mutants by embryo
microinjection of TALEN mRNA. Nature protocols 2013; 8: 2355-2379. 2013/11/02. DOI: 10.1038/nprot.2013.142.
17. Song J, Zhong J, Guo X, et al. Generation of RAG 1- and 2-deficient rabbits by embryo
microinjection of TALENs. Cell research 2013; 23: 1059-1062. 2013/07/10. DOI: 10.1038/cr.2013.85.
18. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in
livestock. Proc Natl Acad Sci U S A 2012; 109: 17382-17387. 2012/10/03. DOI: 10.1073/pnas.1211446109.
19. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited
zygotes. Sci Rep 2013; 3: 2847. 2013/10/11. DOI: 10.1038/srep02847. 20. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous
recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 2015; 5: 10482. 2015/05/23. DOI: 10.1038/srep10482. 21. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle.
Transgenic research 2015; 24: 147-153. 2014/09/11. DOI: 10.1007/s11248-014-9832- x. 22. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and
cynomolgus monkeys. Cell Stem Cell 2014; 14: 323-328. 2014/02/18. DOI: 10.1016/j.stem.2014.01.018.
23. Bassett AR, Tibbit C, Ponting CP, et al. Highly efficient targeted mutagenesis of
Drosophila with the CRISPR/Cas9 system. Cell Rep 2013; 4: 220-228. 2013/07/06. DOI: 10.1016/j.celrep.2013.06.020.
24. Friedland AE, Tzur YB, Esvelt KM, et al. Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nature methods 2013; 10: 741-743. 2013/07/03. DOI: 10.1038/nmeth.2532.
25. Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-
Cas system. Nat Biotechnol 2013; 31: 227-229. 2013/01/31. DOI: 10.1038/nbt.2501. 26. Nakayama T, Fish MB, Fisher M, et al. Simple and efficient CRISPR/Cas9-mediated
targeted mutagenesis in Xenopus tropicalis. Genesis 2013; 51: 835-843. 2013/10/15. DOI: 10.1002/dvg.22720.
27. Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of
multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 2013; 31: 684-686. 2013/08/10. DOI: 10.1038/nbt.2652.
28. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9
nucleases. J Mol Cell Biol 2014; 6: 97-99. 2014/01/10. DOI: 10.1093/jmcb/mjt047. 29. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection
of CRISPR/Cas system. Cell research 2014; 24: 372-375. 2014/02/01. DOI: 10.1038/cr.2014.11.
30. Bevacqua RJ, Fernandez-Martin R, Savy V, et al. Efficient edition of the bovine PRNP
prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology 2016; 86: 1886-1896 e1881. 2016/08/28. DOI: 10.1016/j.theriogenology.2016.06.010.
31. Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and
FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 2015; 5: 13878. 2015/09/12. DOI: 10.1038/srep13878.
32. Crispo M, Mulet AP, Tesson L, et al. Efficient Generation of Myostatin Knock-Out
Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PloS one 2015; 10: e0136690. 2015/08/26. DOI: 10.1371/journal.pone.0136690.
33. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via
Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156: 836-843. 2014/02/04. DOI: 10.1016/j.cell.2014.01.027.
34. Zhang Y, Qin W, Lu X, et al. Programmable base editing of zebrafish genome using a
modified CRISPR-Cas9 system. Nat Commun 2017; 8: 118. 2017/07/26. DOI: 10.1038/s41467-017-00175-6.
35. Park DS, Yoon M, Kweon J, et al. Targeted Base Editing via RNA-Guided Cytidine
Deaminases in Xenopus laevis Embryos. Mol Cells 2017; 40: 823-827. 2017/11/28. DOI: 10.14348/molcells.2017.0262.
36. Kim K, Ryu SM, Kim ST, et al. Highly efficient RNA-guided base editing in mouse
embryos. Nat Biotechnol 2017; 35: 435-437. 2017/03/01. DOI: 10.1038/nbt.3816. 37. Ma Y, Yu L, Zhang X, et al. Highly efficient and precise base editing by engineered
dCas9-guide tRNA adenosine deaminase in rats. Cell Discov 2018; 4: 39. 2018/07/25. DOI: 10.1038/s41421-018-0047-9.
38. Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors
in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 2018; 9: 814-819. 2018/08/02. DOI: 10.1007/s13238-018-0568-x. 39. Liu Z, Chen M, Chen S, et al. Highly efficient RNA-guided base editing in rabbit. Nat
Commun 2018; 9: 2717. 2018/07/15. DOI: 10.1038/s41467-018-05232-2. 40. Xie J, Ge W, Li N, et al. Efficient base editing for multiple genes and loci in pigs using
base editors. Nat Commun 2019; 10: 2852. 2019/06/30. DOI: 10.1038/s41467-019- 10421-8.
41. Li G, Zhou S, Li C, et al. Base pair editing in goat: nonsense codon introgression into
FGF5 results in longer hair. The FEBS journal 2019; 286: 4675-4692. 2019/07/06. DOI: 10.1111/febs.14983.
42. Zhou S, Cai B, He C, et al. Programmable Base Editing of the Sheep Genome Revealed
No Genome-Wide Off-Target Mutations. Front Genet 2019; 10: 215. 2019/04/02. DOI: 10.3389/fgene.2019.00215.
43. Zhang W, Aida T, Del Rosario RCH, et al. Multiplex precise base editing in cynomolgus
monkeys. Nat Commun 2020; 11: 2325. 2020/05/13. DOI: 10.1038/s41467-020- 16173-0.
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[D06D0WM 7/84?/0W 8737W H01,6 H0 Supplementary material for
History of genome editing - from meganucleases to CRISPR Simon E. Tröder and Branko Zevnik
Table S1. Supplementary references for in vivo genome editing Method Species Reference
ZFN mouse 1, 2 rat 3 rabbit 4 pig 5, 6 cattle 7, 8 TALEN
worm 9 zebrafish 10, 11
frog 12 rat 13 mouse 14-16 rabbit 17 pig 18, 19
goat 20 sheep 21
cattle 21
non-human primate 22
CRISPR/Cas9
fly 23
worm 24
zebrafish 25
frog 26
rat 27
rabbit 28
pig 29
cattle 30
goat 31
sheep 32
non-human primate 33
CRISPR base editing
zebrafish 34
frog 35
mouse 36
rat 37, 38
rabbit 39
pig 40
goat 41
sheep 42
non-human primate 43
Supplementary references
1. Meyer M, de Angelis MH, Wurst W, et al. Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A 2010; 107: 15022-15026. 2010/08/06. DOI: 10.1073/pnas.1009424107. 2. Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using
zinc-finger nucleases. Genetics 2010; 186: 451-459. 2010/07/16. DOI: 10.1534/genetics.110.117002.
3. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of
zinc-finger nucleases. Science 2009; 325: 433. 2009/07/25. DOI: 10.1126/science.1172447.
4. Flisikowska T, Thorey IS, Offner S, et al. Efficient immunoglobulin gene disruption
and targeted replacement in rabbit using zinc finger nucleases. PloS one 2011; 6: e21045. 2011/06/23. DOI: 10.1371/journal.pone.0021045. 5. Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout
in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12017. 2011/07/07. DOI: 10.1073/pnas.1106422108.
6. Whyte JJ, Zhao J, Wells KD, et al. Gene targeting with zinc finger nucleases to
produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78: 2. 2011/01/27. DOI: 10.1002/mrd.21271.
7. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows. Nat Commun 2013; 4: 2565. 2013/10/15. DOI: 10.1038/ncomms3565.
8. Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG)
gene via zinc-finger nucleases in cattle. Cell research 2011; 21: 1638-1640. 2011/09/14. DOI: 10.1038/cr.2011.153.
9. Wood AJ, Lo TW, Zeitler B, et al. Targeted genome editing across species using ZFNs
and TALENs. Science 2011; 333: 307. 2011/06/28. DOI: 10.1126/science.1207773. 10. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using
customized TALENs. Nat Biotechnol 2011; 29: 699-700. 2011/08/09. DOI: 10.1038/nbt.1939.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish
cells using engineered TALENs. Nat Biotechnol 2011; 29: 697-698. 2011/08/09. DOI: 10.1038/nbt.1934.
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using
engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012; 109: 17484-17489. 2012/10/10. DOI: 10.1073/pnas.1215421109. 13. Tesson L, Usal C, Menoret S, et al. Knockout rats generated by embryo microinjection
of TALENs. Nat Biotechnol 2011; 29: 695-696. 2011/08/09. DOI: 10.1038/nbt.1940. 14. Panda SK, Wefers B, Ortiz O, et al. Highly efficient targeted mutagenesis in mice
using TALENs. Genetics 2013; 195: 703-713. 2013/08/28. DOI: 10.1534/genetics.113.156570.
15. Sung YH, Baek IJ, Kim DH, et al. Knockout mice created by TALEN-mediated gene
targeting. Nat Biotechnol 2013; 31: 23-24. 2013/01/11. DOI: 10.1038/nbt.2477. 16. Wefers B, Panda SK, Ortiz O, et al. Generation of targeted mouse mutants by embryo
microinjection of TALEN mRNA. Nature protocols 2013; 8: 2355-2379. 2013/11/02. DOI: 10.1038/nprot.2013.142.
17. Song J, Zhong J, Guo X, et al. Generation of RAG 1- and 2-deficient rabbits by embryo
microinjection of TALENs. Cell research 2013; 23: 1059-1062. 2013/07/10. DOI: 10.1038/cr.2013.85.
18. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in
livestock. Proc Natl Acad Sci U S A 2012; 109: 17382-17387. 2012/10/03. DOI: 10.1073/pnas.1211446109.
19. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited
zygotes. Sci Rep 2013; 3: 2847. 2013/10/11. DOI: 10.1038/srep02847. 20. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous
recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 2015; 5: 10482. 2015/05/23. DOI: 10.1038/srep10482. 21. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle.
Transgenic research 2015; 24: 147-153. 2014/09/11. DOI: 10.1007/s11248-014-9832- x. 22. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and
cynomolgus monkeys. Cell Stem Cell 2014; 14: 323-328. 2014/02/18. DOI: 10.1016/j.stem.2014.01.018.
23. Bassett AR, Tibbit C, Ponting CP, et al. Highly efficient targeted mutagenesis of
Drosophila with the CRISPR/Cas9 system. Cell Rep 2013; 4: 220-228. 2013/07/06. DOI: 10.1016/j.celrep.2013.06.020.
24. Friedland AE, Tzur YB, Esvelt KM, et al. Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nature methods 2013; 10: 741-743. 2013/07/03. DOI: 10.1038/nmeth.2532.
25. Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-
Cas system. Nat Biotechnol 2013; 31: 227-229. 2013/01/31. DOI: 10.1038/nbt.2501. 26. Nakayama T, Fish MB, Fisher M, et al. Simple and efficient CRISPR/Cas9-mediated
targeted mutagenesis in Xenopus tropicalis. Genesis 2013; 51: 835-843. 2013/10/15. DOI: 10.1002/dvg.22720.
27. Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of
multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 2013; 31: 684-686. 2013/08/10. DOI: 10.1038/nbt.2652.
28. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9
nucleases. J Mol Cell Biol 2014; 6: 97-99. 2014/01/10. DOI: 10.1093/jmcb/mjt047. 29. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection
of CRISPR/Cas system. Cell research 2014; 24: 372-375. 2014/02/01. DOI: 10.1038/cr.2014.11.
30. Bevacqua RJ, Fernandez-Martin R, Savy V, et al. Efficient edition of the bovine PRNP
prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology 2016; 86: 1886-1896 e1881. 2016/08/28. DOI: 10.1016/j.theriogenology.2016.06.010.
31. Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and
FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 2015; 5: 13878. 2015/09/12. DOI: 10.1038/srep13878.
32. Crispo M, Mulet AP, Tesson L, et al. Efficient Generation of Myostatin Knock-Out
Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PloS one 2015; 10: e0136690. 2015/08/26. DOI: 10.1371/journal.pone.0136690.
33. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via
Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156: 836-843. 2014/02/04. DOI: 10.1016/j.cell.2014.01.027.
34. Zhang Y, Qin W, Lu X, et al. Programmable base editing of zebrafish genome using a
modified CRISPR-Cas9 system. Nat Commun 2017; 8: 118. 2017/07/26. DOI: 10.1038/s41467-017-00175-6.
35. Park DS, Yoon M, Kweon J, et al. Targeted Base Editing via RNA-Guided Cytidine
Deaminases in Xenopus laevis Embryos. Mol Cells 2017; 40: 823-827. 2017/11/28. DOI: 10.14348/molcells.2017.0262.
36. Kim K, Ryu SM, Kim ST, et al. Highly efficient RNA-guided base editing in mouse
embryos. Nat Biotechnol 2017; 35: 435-437. 2017/03/01. DOI: 10.1038/nbt.3816. 37. Ma Y, Yu L, Zhang X, et al. Highly efficient and precise base editing by engineered
dCas9-guide tRNA adenosine deaminase in rats. Cell Discov 2018; 4: 39. 2018/07/25. DOI: 10.1038/s41421-018-0047-9.
38. Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors
in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 2018; 9: 814-819. 2018/08/02. DOI: 10.1007/s13238-018-0568-x. 39. Liu Z, Chen M, Chen S, et al. Highly efficient RNA-guided base editing in rabbit. Nat
Commun 2018; 9: 2717. 2018/07/15. DOI: 10.1038/s41467-018-05232-2. 40. Xie J, Ge W, Li N, et al. Efficient base editing for multiple genes and loci in pigs using
base editors. Nat Commun 2019; 10: 2852. 2019/06/30. DOI: 10.1038/s41467-019- 10421-8.
41. Li G, Zhou S, Li C, et al. Base pair editing in goat: nonsense codon introgression into
FGF5 results in longer hair. The FEBS journal 2019; 286: 4675-4692. 2019/07/06. DOI: 10.1111/febs.14983.
42. Zhou S, Cai B, He C, et al. Programmable Base Editing of the Sheep Genome Revealed
No Genome-Wide Off-Target Mutations. Front Genet 2019; 10: 215. 2019/04/02. DOI: 10.3389/fgene.2019.00215.
43. Zhang W, Aida T, Del Rosario RCH, et al. Multiplex precise base editing in cynomolgus
monkeys. Nat Commun 2020; 11: 2325. 2020/05/13. DOI: 10.1038/s41467-020- 16173-0.
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[D06D0WM 7/84?/0W 8737W H01,6 H0 Supplementary material for
History of genome editing - from meganucleases to CRISPR Simon E. Tröder and Branko Zevnik
Table S1. Supplementary references for in vivo genome editing Method Species Reference
ZFN mouse 1, 2 rat 3 rabbit 4 pig 5, 6 cattle 7, 8 TALEN
worm 9 zebrafish 10, 11
frog 12 rat 13 mouse 14-16 rabbit 17 pig 18, 19
goat 20 sheep 21
cattle 21
non-human primate 22
CRISPR/Cas9
fly 23
worm 24
zebrafish 25
frog 26
rat 27
rabbit 28
pig 29
cattle 30
goat 31
sheep 32
non-human primate 33
CRISPR base editing
zebrafish 34
frog 35
mouse 36
rat 37, 38
rabbit 39
pig 40
goat 41
sheep 42
non-human primate 43
Supplementary references
1. Meyer M, de Angelis MH, Wurst W, et al. Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A 2010; 107: 15022-15026. 2010/08/06. DOI: 10.1073/pnas.1009424107. 2. Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using
zinc-finger nucleases. Genetics 2010; 186: 451-459. 2010/07/16. DOI: 10.1534/genetics.110.117002.
3. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of
zinc-finger nucleases. Science 2009; 325: 433. 2009/07/25. DOI: 10.1126/science.1172447.
4. Flisikowska T, Thorey IS, Offner S, et al. Efficient immunoglobulin gene disruption
and targeted replacement in rabbit using zinc finger nucleases. PloS one 2011; 6: e21045. 2011/06/23. DOI: 10.1371/journal.pone.0021045. 5. Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout
in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12017. 2011/07/07. DOI: 10.1073/pnas.1106422108.
6. Whyte JJ, Zhao J, Wells KD, et al. Gene targeting with zinc finger nucleases to
produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78: 2. 2011/01/27. DOI: 10.1002/mrd.21271.
7. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows. Nat Commun 2013; 4: 2565. 2013/10/15. DOI: 10.1038/ncomms3565.
8. Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG)
gene via zinc-finger nucleases in cattle. Cell research 2011; 21: 1638-1640. 2011/09/14. DOI: 10.1038/cr.2011.153.
9. Wood AJ, Lo TW, Zeitler B, et al. Targeted genome editing across species using ZFNs
and TALENs. Science 2011; 333: 307. 2011/06/28. DOI: 10.1126/science.1207773. 10. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using
customized TALENs. Nat Biotechnol 2011; 29: 699-700. 2011/08/09. DOI: 10.1038/nbt.1939.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish
cells using engineered TALENs. Nat Biotechnol 2011; 29: 697-698. 2011/08/09. DOI: 10.1038/nbt.1934.
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using
engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012; 109: 17484-17489. 2012/10/10. DOI: 10.1073/pnas.1215421109. 13. Tesson L, Usal C, Menoret S, et al. Knockout rats generated by embryo microinjection
of TALENs. Nat Biotechnol 2011; 29: 695-696. 2011/08/09. DOI: 10.1038/nbt.1940. 14. Panda SK, Wefers B, Ortiz O, et al. Highly efficient targeted mutagenesis in mice
using TALENs. Genetics 2013; 195: 703-713. 2013/08/28. DOI: 10.1534/genetics.113.156570.
15. Sung YH, Baek IJ, Kim DH, et al. Knockout mice created by TALEN-mediated gene
targeting. Nat Biotechnol 2013; 31: 23-24. 2013/01/11. DOI: 10.1038/nbt.2477. 16. Wefers B, Panda SK, Ortiz O, et al. Generation of targeted mouse mutants by embryo
microinjection of TALEN mRNA. Nature protocols 2013; 8: 2355-2379. 2013/11/02. DOI: 10.1038/nprot.2013.142.
17. Song J, Zhong J, Guo X, et al. Generation of RAG 1- and 2-deficient rabbits by embryo
microinjection of TALENs. Cell research 2013; 23: 1059-1062. 2013/07/10. DOI: 10.1038/cr.2013.85.
18. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in
livestock. Proc Natl Acad Sci U S A 2012; 109: 17382-17387. 2012/10/03. DOI: 10.1073/pnas.1211446109.
19. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited
zygotes. Sci Rep 2013; 3: 2847. 2013/10/11. DOI: 10.1038/srep02847. 20. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous
recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 2015; 5: 10482. 2015/05/23. DOI: 10.1038/srep10482. 21. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle.
Transgenic research 2015; 24: 147-153. 2014/09/11. DOI: 10.1007/s11248-014-9832- x. 22. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and
cynomolgus monkeys. Cell Stem Cell 2014; 14: 323-328. 2014/02/18. DOI: 10.1016/j.stem.2014.01.018.
23. Bassett AR, Tibbit C, Ponting CP, et al. Highly efficient targeted mutagenesis of
Drosophila with the CRISPR/Cas9 system. Cell Rep 2013; 4: 220-228. 2013/07/06. DOI: 10.1016/j.celrep.2013.06.020.
24. Friedland AE, Tzur YB, Esvelt KM, et al. Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nature methods 2013; 10: 741-743. 2013/07/03. DOI: 10.1038/nmeth.2532.
25. Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-
Cas system. Nat Biotechnol 2013; 31: 227-229. 2013/01/31. DOI: 10.1038/nbt.2501. 26. Nakayama T, Fish MB, Fisher M, et al. Simple and efficient CRISPR/Cas9-mediated
targeted mutagenesis in Xenopus tropicalis. Genesis 2013; 51: 835-843. 2013/10/15. DOI: 10.1002/dvg.22720.
27. Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of
multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 2013; 31: 684-686. 2013/08/10. DOI: 10.1038/nbt.2652.
28. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9
nucleases. J Mol Cell Biol 2014; 6: 97-99. 2014/01/10. DOI: 10.1093/jmcb/mjt047. 29. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection
of CRISPR/Cas system. Cell research 2014; 24: 372-375. 2014/02/01. DOI: 10.1038/cr.2014.11.
30. Bevacqua RJ, Fernandez-Martin R, Savy V, et al. Efficient edition of the bovine PRNP
prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology 2016; 86: 1886-1896 e1881. 2016/08/28. DOI: 10.1016/j.theriogenology.2016.06.010.
31. Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and
FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 2015; 5: 13878. 2015/09/12. DOI: 10.1038/srep13878.
32. Crispo M, Mulet AP, Tesson L, et al. Efficient Generation of Myostatin Knock-Out
Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PloS one 2015; 10: e0136690. 2015/08/26. DOI: 10.1371/journal.pone.0136690.
33. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via
Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156: 836-843. 2014/02/04. DOI: 10.1016/j.cell.2014.01.027.
34. Zhang Y, Qin W, Lu X, et al. Programmable base editing of zebrafish genome using a
modified CRISPR-Cas9 system. Nat Commun 2017; 8: 118. 2017/07/26. DOI: 10.1038/s41467-017-00175-6.
35. Park DS, Yoon M, Kweon J, et al. Targeted Base Editing via RNA-Guided Cytidine
Deaminases in Xenopus laevis Embryos. Mol Cells 2017; 40: 823-827. 2017/11/28. DOI: 10.14348/molcells.2017.0262.
36. Kim K, Ryu SM, Kim ST, et al. Highly efficient RNA-guided base editing in mouse
embryos. Nat Biotechnol 2017; 35: 435-437. 2017/03/01. DOI: 10.1038/nbt.3816. 37. Ma Y, Yu L, Zhang X, et al. Highly efficient and precise base editing by engineered
dCas9-guide tRNA adenosine deaminase in rats. Cell Discov 2018; 4: 39. 2018/07/25. DOI: 10.1038/s41421-018-0047-9.
38. Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors
in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 2018; 9: 814-819. 2018/08/02. DOI: 10.1007/s13238-018-0568-x. 39. Liu Z, Chen M, Chen S, et al. Highly efficient RNA-guided base editing in rabbit. Nat
Commun 2018; 9: 2717. 2018/07/15. DOI: 10.1038/s41467-018-05232-2. 40. Xie J, Ge W, Li N, et al. Efficient base editing for multiple genes and loci in pigs using
base editors. Nat Commun 2019; 10: 2852. 2019/06/30. DOI: 10.1038/s41467-019- 10421-8.
41. Li G, Zhou S, Li C, et al. Base pair editing in goat: nonsense codon introgression into
FGF5 results in longer hair. The FEBS journal 2019; 286: 4675-4692. 2019/07/06. DOI: 10.1111/febs.14983.
42. Zhou S, Cai B, He C, et al. Programmable Base Editing of the Sheep Genome Revealed
No Genome-Wide Off-Target Mutations. Front Genet 2019; 10: 215. 2019/04/02. DOI: 10.3389/fgene.2019.00215.
43. Zhang W, Aida T, Del Rosario RCH, et al. Multiplex precise base editing in cynomolgus
monkeys. Nat Commun 2020; 11: 2325. 2020/05/13. DOI: 10.1038/s41467-020- 16173-0.
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[D06D0WM 7/84?/0W 8737W H01,6 H0 Supplementary material for
History of genome editing - from meganucleases to CRISPR Simon E. Tröder and Branko Zevnik
Table S1. Supplementary references for in vivo genome editing Method Species Reference
ZFN mouse 1, 2 rat 3 rabbit 4 pig 5, 6 cattle 7, 8 TALEN
worm 9 zebrafish 10, 11
frog 12 rat 13 mouse 14-16 rabbit 17 pig 18, 19
goat 20 sheep 21
cattle 21
non-human primate 22
CRISPR/Cas9
fly 23
worm 24
zebrafish 25
frog 26
rat 27
rabbit 28
pig 29
cattle 30
goat 31
sheep 32
non-human primate 33
CRISPR base editing
zebrafish 34
frog 35
mouse 36
rat 37, 38
rabbit 39
pig 40
goat 41
sheep 42
non-human primate 43
Supplementary references
1. Meyer M, de Angelis MH, Wurst W, et al. Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A 2010; 107: 15022-15026. 2010/08/06. DOI: 10.1073/pnas.1009424107. 2. Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using
zinc-finger nucleases. Genetics 2010; 186: 451-459. 2010/07/16. DOI: 10.1534/genetics.110.117002.
3. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of
zinc-finger nucleases. Science 2009; 325: 433. 2009/07/25. DOI: 10.1126/science.1172447.
4. Flisikowska T, Thorey IS, Offner S, et al. Efficient immunoglobulin gene disruption
and targeted replacement in rabbit using zinc finger nucleases. PloS one 2011; 6: e21045. 2011/06/23. DOI: 10.1371/journal.pone.0021045. 5. Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout
in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12017. 2011/07/07. DOI: 10.1073/pnas.1106422108.
6. Whyte JJ, Zhao J, Wells KD, et al. Gene targeting with zinc finger nucleases to
produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78: 2. 2011/01/27. DOI: 10.1002/mrd.21271.
7. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows. Nat Commun 2013; 4: 2565. 2013/10/15. DOI: 10.1038/ncomms3565.
8. Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG)
gene via zinc-finger nucleases in cattle. Cell research 2011; 21: 1638-1640. 2011/09/14. DOI: 10.1038/cr.2011.153.
9. Wood AJ, Lo TW, Zeitler B, et al. Targeted genome editing across species using ZFNs
and TALENs. Science 2011; 333: 307. 2011/06/28. DOI: 10.1126/science.1207773. 10. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using
customized TALENs. Nat Biotechnol 2011; 29: 699-700. 2011/08/09. DOI: 10.1038/nbt.1939.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish
cells using engineered TALENs. Nat Biotechnol 2011; 29: 697-698. 2011/08/09. DOI: 10.1038/nbt.1934.
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using
engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012; 109: 17484-17489. 2012/10/10. DOI: 10.1073/pnas.1215421109. 13. Tesson L, Usal C, Menoret S, et al. Knockout rats generated by embryo microinjection
of TALENs. Nat Biotechnol 2011; 29: 695-696. 2011/08/09. DOI: 10.1038/nbt.1940. 14. Panda SK, Wefers B, Ortiz O, et al. Highly efficient targeted mutagenesis in mice
using TALENs. Genetics 2013; 195: 703-713. 2013/08/28. DOI: 10.1534/genetics.113.156570.
15. Sung YH, Baek IJ, Kim DH, et al. Knockout mice created by TALEN-mediated gene
targeting. Nat Biotechnol 2013; 31: 23-24. 2013/01/11. DOI: 10.1038/nbt.2477. 16. Wefers B, Panda SK, Ortiz O, et al. Generation of targeted mouse mutants by embryo
microinjection of TALEN mRNA. Nature protocols 2013; 8: 2355-2379. 2013/11/02. DOI: 10.1038/nprot.2013.142.
17. Song J, Zhong J, Guo X, et al. Generation of RAG 1- and 2-deficient rabbits by embryo
microinjection of TALENs. Cell research 2013; 23: 1059-1062. 2013/07/10. DOI: 10.1038/cr.2013.85.
18. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in
livestock. Proc Natl Acad Sci U S A 2012; 109: 17382-17387. 2012/10/03. DOI: 10.1073/pnas.1211446109.
19. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited
zygotes. Sci Rep 2013; 3: 2847. 2013/10/11. DOI: 10.1038/srep02847. 20. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous
recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 2015; 5: 10482. 2015/05/23. DOI: 10.1038/srep10482. 21. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle.
Transgenic research 2015; 24: 147-153. 2014/09/11. DOI: 10.1007/s11248-014-9832- x. 22. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and
cynomolgus monkeys. Cell Stem Cell 2014; 14: 323-328. 2014/02/18. DOI: 10.1016/j.stem.2014.01.018.
23. Bassett AR, Tibbit C, Ponting CP, et al. Highly efficient targeted mutagenesis of
Drosophila with the CRISPR/Cas9 system. Cell Rep 2013; 4: 220-228. 2013/07/06. DOI: 10.1016/j.celrep.2013.06.020.
24. Friedland AE, Tzur YB, Esvelt KM, et al. Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nature methods 2013; 10: 741-743. 2013/07/03. DOI: 10.1038/nmeth.2532.
25. Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-
Cas system. Nat Biotechnol 2013; 31: 227-229. 2013/01/31. DOI: 10.1038/nbt.2501. 26. Nakayama T, Fish MB, Fisher M, et al. Simple and efficient CRISPR/Cas9-mediated
targeted mutagenesis in Xenopus tropicalis. Genesis 2013; 51: 835-843. 2013/10/15. DOI: 10.1002/dvg.22720.
27. Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of
multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 2013; 31: 684-686. 2013/08/10. DOI: 10.1038/nbt.2652.
28. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9
nucleases. J Mol Cell Biol 2014; 6: 97-99. 2014/01/10. DOI: 10.1093/jmcb/mjt047. 29. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection
of CRISPR/Cas system. Cell research 2014; 24: 372-375. 2014/02/01. DOI: 10.1038/cr.2014.11.
30. Bevacqua RJ, Fernandez-Martin R, Savy V, et al. Efficient edition of the bovine PRNP
prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology 2016; 86: 1886-1896 e1881. 2016/08/28. DOI: 10.1016/j.theriogenology.2016.06.010.
31. Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and
FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 2015; 5: 13878. 2015/09/12. DOI: 10.1038/srep13878.
32. Crispo M, Mulet AP, Tesson L, et al. Efficient Generation of Myostatin Knock-Out
Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PloS one 2015; 10: e0136690. 2015/08/26. DOI: 10.1371/journal.pone.0136690.
33. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via
Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156: 836-843. 2014/02/04. DOI: 10.1016/j.cell.2014.01.027.
34. Zhang Y, Qin W, Lu X, et al. Programmable base editing of zebrafish genome using a
modified CRISPR-Cas9 system. Nat Commun 2017; 8: 118. 2017/07/26. DOI: 10.1038/s41467-017-00175-6.
35. Park DS, Yoon M, Kweon J, et al. Targeted Base Editing via RNA-Guided Cytidine
Deaminases in Xenopus laevis Embryos. Mol Cells 2017; 40: 823-827. 2017/11/28. DOI: 10.14348/molcells.2017.0262.
36. Kim K, Ryu SM, Kim ST, et al. Highly efficient RNA-guided base editing in mouse
embryos. Nat Biotechnol 2017; 35: 435-437. 2017/03/01. DOI: 10.1038/nbt.3816. 37. Ma Y, Yu L, Zhang X, et al. Highly efficient and precise base editing by engineered
dCas9-guide tRNA adenosine deaminase in rats. Cell Discov 2018; 4: 39. 2018/07/25. DOI: 10.1038/s41421-018-0047-9.
38. Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors
in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 2018; 9: 814-819. 2018/08/02. DOI: 10.1007/s13238-018-0568-x. 39. Liu Z, Chen M, Chen S, et al. Highly efficient RNA-guided base editing in rabbit. Nat
Commun 2018; 9: 2717. 2018/07/15. DOI: 10.1038/s41467-018-05232-2. 40. Xie J, Ge W, Li N, et al. Efficient base editing for multiple genes and loci in pigs using
base editors. Nat Commun 2019; 10: 2852. 2019/06/30. DOI: 10.1038/s41467-019- 10421-8.
41. Li G, Zhou S, Li C, et al. Base pair editing in goat: nonsense codon introgression into
FGF5 results in longer hair. The FEBS journal 2019; 286: 4675-4692. 2019/07/06. DOI: 10.1111/febs.14983.
42. Zhou S, Cai B, He C, et al. Programmable Base Editing of the Sheep Genome Revealed
No Genome-Wide Off-Target Mutations. Front Genet 2019; 10: 215. 2019/04/02. DOI: 10.3389/fgene.2019.00215.
43. Zhang W, Aida T, Del Rosario RCH, et al. Multiplex precise base editing in cynomolgus
monkeys. Nat Commun 2020; 11: 2325. 2020/05/13. DOI: 10.1038/s41467-020- 16173-0.
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[D06D0WM 7/84?/0W 8737W H01,6 H0 Supplementary material for
History of genome editing - from meganucleases to CRISPR Simon E. Tröder and Branko Zevnik
Table S1. Supplementary references for in vivo genome editing Method Species Reference
ZFN mouse 1, 2 rat 3 rabbit 4 pig 5, 6 cattle 7, 8 TALEN
worm 9 zebrafish 10, 11
frog 12 rat 13 mouse 14-16 rabbit 17 pig 18, 19
goat 20 sheep 21
cattle 21
non-human primate 22
CRISPR/Cas9
fly 23
worm 24
zebrafish 25
frog 26
rat 27
rabbit 28
pig 29
cattle 30
goat 31
sheep 32
non-human primate 33
CRISPR base editing
zebrafish 34
frog 35
mouse 36
rat 37, 38
rabbit 39
pig 40
goat 41
sheep 42
non-human primate 43
Supplementary references
1. Meyer M, de Angelis MH, Wurst W, et al. Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A 2010; 107: 15022-15026. 2010/08/06. DOI: 10.1073/pnas.1009424107. 2. Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using
zinc-finger nucleases. Genetics 2010; 186: 451-459. 2010/07/16. DOI: 10.1534/genetics.110.117002.
3. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of
zinc-finger nucleases. Science 2009; 325: 433. 2009/07/25. DOI: 10.1126/science.1172447.
4. Flisikowska T, Thorey IS, Offner S, et al. Efficient immunoglobulin gene disruption
and targeted replacement in rabbit using zinc finger nucleases. PloS one 2011; 6: e21045. 2011/06/23. DOI: 10.1371/journal.pone.0021045. 5. Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout
in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12017. 2011/07/07. DOI: 10.1073/pnas.1106422108.
6. Whyte JJ, Zhao J, Wells KD, et al. Gene targeting with zinc finger nucleases to
produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78: 2. 2011/01/27. DOI: 10.1002/mrd.21271.
7. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows. Nat Commun 2013; 4: 2565. 2013/10/15. DOI: 10.1038/ncomms3565.
8. Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG)
gene via zinc-finger nucleases in cattle. Cell research 2011; 21: 1638-1640. 2011/09/14. DOI: 10.1038/cr.2011.153.
9. Wood AJ, Lo TW, Zeitler B, et al. Targeted genome editing across species using ZFNs
and TALENs. Science 2011; 333: 307. 2011/06/28. DOI: 10.1126/science.1207773. 10. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using
customized TALENs. Nat Biotechnol 2011; 29: 699-700. 2011/08/09. DOI: 10.1038/nbt.1939.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish
cells using engineered TALENs. Nat Biotechnol 2011; 29: 697-698. 2011/08/09. DOI: 10.1038/nbt.1934.
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using
engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012; 109: 17484-17489. 2012/10/10. DOI: 10.1073/pnas.1215421109. 13. Tesson L, Usal C, Menoret S, et al. Knockout rats generated by embryo microinjection
of TALENs. Nat Biotechnol 2011; 29: 695-696. 2011/08/09. DOI: 10.1038/nbt.1940. 14. Panda SK, Wefers B, Ortiz O, et al. Highly efficient targeted mutagenesis in mice
using TALENs. Genetics 2013; 195: 703-713. 2013/08/28. DOI: 10.1534/genetics.113.156570.
15. Sung YH, Baek IJ, Kim DH, et al. Knockout mice created by TALEN-mediated gene
targeting. Nat Biotechnol 2013; 31: 23-24. 2013/01/11. DOI: 10.1038/nbt.2477. 16. Wefers B, Panda SK, Ortiz O, et al. Generation of targeted mouse mutants by embryo
microinjection of TALEN mRNA. Nature protocols 2013; 8: 2355-2379. 2013/11/02. DOI: 10.1038/nprot.2013.142.
17. Song J, Zhong J, Guo X, et al. Generation of RAG 1- and 2-deficient rabbits by embryo
microinjection of TALENs. Cell research 2013; 23: 1059-1062. 2013/07/10. DOI: 10.1038/cr.2013.85.
18. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in
livestock. Proc Natl Acad Sci U S A 2012; 109: 17382-17387. 2012/10/03. DOI: 10.1073/pnas.1211446109.
19. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited
zygotes. Sci Rep 2013; 3: 2847. 2013/10/11. DOI: 10.1038/srep02847. 20. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous
recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 2015; 5: 10482. 2015/05/23. DOI: 10.1038/srep10482. 21. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle.
Transgenic research 2015; 24: 147-153. 2014/09/11. DOI: 10.1007/s11248-014-9832- x. 22. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and
cynomolgus monkeys. Cell Stem Cell 2014; 14: 323-328. 2014/02/18. DOI: 10.1016/j.stem.2014.01.018.
23. Bassett AR, Tibbit C, Ponting CP, et al. Highly efficient targeted mutagenesis of
Drosophila with the CRISPR/Cas9 system. Cell Rep 2013; 4: 220-228. 2013/07/06. DOI: 10.1016/j.celrep.2013.06.020.
24. Friedland AE, Tzur YB, Esvelt KM, et al. Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nature methods 2013; 10: 741-743. 2013/07/03. DOI: 10.1038/nmeth.2532.
25. Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-
Cas system. Nat Biotechnol 2013; 31: 227-229. 2013/01/31. DOI: 10.1038/nbt.2501. 26. Nakayama T, Fish MB, Fisher M, et al. Simple and efficient CRISPR/Cas9-mediated
targeted mutagenesis in Xenopus tropicalis. Genesis 2013; 51: 835-843. 2013/10/15. DOI: 10.1002/dvg.22720.
27. Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of
multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 2013; 31: 684-686. 2013/08/10. DOI: 10.1038/nbt.2652.
28. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9
nucleases. J Mol Cell Biol 2014; 6: 97-99. 2014/01/10. DOI: 10.1093/jmcb/mjt047. 29. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection
of CRISPR/Cas system. Cell research 2014; 24: 372-375. 2014/02/01. DOI: 10.1038/cr.2014.11.
30. Bevacqua RJ, Fernandez-Martin R, Savy V, et al. Efficient edition of the bovine PRNP
prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology 2016; 86: 1886-1896 e1881. 2016/08/28. DOI: 10.1016/j.theriogenology.2016.06.010.
31. Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and
FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 2015; 5: 13878. 2015/09/12. DOI: 10.1038/srep13878.
32. Crispo M, Mulet AP, Tesson L, et al. Efficient Generation of Myostatin Knock-Out
Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PloS one 2015; 10: e0136690. 2015/08/26. DOI: 10.1371/journal.pone.0136690.
33. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via
Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156: 836-843. 2014/02/04. DOI: 10.1016/j.cell.2014.01.027.
34. Zhang Y, Qin W, Lu X, et al. Programmable base editing of zebrafish genome using a
modified CRISPR-Cas9 system. Nat Commun 2017; 8: 118. 2017/07/26. DOI: 10.1038/s41467-017-00175-6.
35. Park DS, Yoon M, Kweon J, et al. Targeted Base Editing via RNA-Guided Cytidine
Deaminases in Xenopus laevis Embryos. Mol Cells 2017; 40: 823-827. 2017/11/28. DOI: 10.14348/molcells.2017.0262.
36. Kim K, Ryu SM, Kim ST, et al. Highly efficient RNA-guided base editing in mouse
embryos. Nat Biotechnol 2017; 35: 435-437. 2017/03/01. DOI: 10.1038/nbt.3816. 37. Ma Y, Yu L, Zhang X, et al. Highly efficient and precise base editing by engineered
dCas9-guide tRNA adenosine deaminase in rats. Cell Discov 2018; 4: 39. 2018/07/25. DOI: 10.1038/s41421-018-0047-9.
38. Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors
in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 2018; 9: 814-819. 2018/08/02. DOI: 10.1007/s13238-018-0568-x. 39. Liu Z, Chen M, Chen S, et al. Highly efficient RNA-guided base editing in rabbit. Nat
Commun 2018; 9: 2717. 2018/07/15. DOI: 10.1038/s41467-018-05232-2. 40. Xie J, Ge W, Li N, et al. Efficient base editing for multiple genes and loci in pigs using
base editors. Nat Commun 2019; 10: 2852. 2019/06/30. DOI: 10.1038/s41467-019- 10421-8.
41. Li G, Zhou S, Li C, et al. Base pair editing in goat: nonsense codon introgression into
FGF5 results in longer hair. The FEBS journal 2019; 286: 4675-4692. 2019/07/06. DOI: 10.1111/febs.14983.
42. Zhou S, Cai B, He C, et al. Programmable Base Editing of the Sheep Genome Revealed
No Genome-Wide Off-Target Mutations. Front Genet 2019; 10: 215. 2019/04/02. DOI: 10.3389/fgene.2019.00215.
43. Zhang W, Aida T, Del Rosario RCH, et al. Multiplex precise base editing in cynomolgus
monkeys. Nat Commun 2020; 11: 2325. 2020/05/13. DOI: 10.1038/s41467-020- 16173-0.
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[D06D0WM 7/84?/0W 8737W H01,6 H0 Supplementary material for
History of genome editing - from meganucleases to CRISPR Simon E. Tröder and Branko Zevnik
Table S1. Supplementary references for in vivo genome editing Method Species Reference
ZFN mouse 1, 2 rat 3 rabbit 4 pig 5, 6 cattle 7, 8 TALEN
worm 9 zebrafish 10, 11
frog 12 rat 13 mouse 14-16 rabbit 17 pig 18, 19
goat 20 sheep 21
cattle 21
non-human primate 22
CRISPR/Cas9
fly 23
worm 24
zebrafish 25
frog 26
rat 27
rabbit 28
pig 29
cattle 30
goat 31
sheep 32
non-human primate 33
CRISPR base editing
zebrafish 34
frog 35
mouse 36
rat 37, 38
rabbit 39
pig 40
goat 41
sheep 42
non-human primate 43
Supplementary references
1. Meyer M, de Angelis MH, Wurst W, et al. Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A 2010; 107: 15022-15026. 2010/08/06. DOI: 10.1073/pnas.1009424107. 2. Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using
zinc-finger nucleases. Genetics 2010; 186: 451-459. 2010/07/16. DOI: 10.1534/genetics.110.117002.
3. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of
zinc-finger nucleases. Science 2009; 325: 433. 2009/07/25. DOI: 10.1126/science.1172447.
4. Flisikowska T, Thorey IS, Offner S, et al. Efficient immunoglobulin gene disruption
and targeted replacement in rabbit using zinc finger nucleases. PloS one 2011; 6: e21045. 2011/06/23. DOI: 10.1371/journal.pone.0021045. 5. Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout
in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12017. 2011/07/07. DOI: 10.1073/pnas.1106422108.
6. Whyte JJ, Zhao J, Wells KD, et al. Gene targeting with zinc finger nucleases to
produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78: 2. 2011/01/27. DOI: 10.1002/mrd.21271.
7. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows. Nat Commun 2013; 4: 2565. 2013/10/15. DOI: 10.1038/ncomms3565.
8. Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG)
gene via zinc-finger nucleases in cattle. Cell research 2011; 21: 1638-1640. 2011/09/14. DOI: 10.1038/cr.2011.153.
9. Wood AJ, Lo TW, Zeitler B, et al. Targeted genome editing across species using ZFNs
and TALENs. Science 2011; 333: 307. 2011/06/28. DOI: 10.1126/science.1207773. 10. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using
customized TALENs. Nat Biotechnol 2011; 29: 699-700. 2011/08/09. DOI: 10.1038/nbt.1939.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish
cells using engineered TALENs. Nat Biotechnol 2011; 29: 697-698. 2011/08/09. DOI: 10.1038/nbt.1934.
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using
engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012; 109: 17484-17489. 2012/10/10. DOI: 10.1073/pnas.1215421109. 13. Tesson L, Usal C, Menoret S, et al. Knockout rats generated by embryo microinjection
of TALENs. Nat Biotechnol 2011; 29: 695-696. 2011/08/09. DOI: 10.1038/nbt.1940. 14. Panda SK, Wefers B, Ortiz O, et al. Highly efficient targeted mutagenesis in mice
using TALENs. Genetics 2013; 195: 703-713. 2013/08/28. DOI: 10.1534/genetics.113.156570.
15. Sung YH, Baek IJ, Kim DH, et al. Knockout mice created by TALEN-mediated gene
targeting. Nat Biotechnol 2013; 31: 23-24. 2013/01/11. DOI: 10.1038/nbt.2477. 16. Wefers B, Panda SK, Ortiz O, et al. Generation of targeted mouse mutants by embryo
microinjection of TALEN mRNA. Nature protocols 2013; 8: 2355-2379. 2013/11/02. DOI: 10.1038/nprot.2013.142.
17. Song J, Zhong J, Guo X, et al. Generation of RAG 1- and 2-deficient rabbits by embryo
microinjection of TALENs. Cell research 2013; 23: 1059-1062. 2013/07/10. DOI: 10.1038/cr.2013.85.
18. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in
livestock. Proc Natl Acad Sci U S A 2012; 109: 17382-17387. 2012/10/03. DOI: 10.1073/pnas.1211446109.
19. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited
zygotes. Sci Rep 2013; 3: 2847. 2013/10/11. DOI: 10.1038/srep02847. 20. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous
recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 2015; 5: 10482. 2015/05/23. DOI: 10.1038/srep10482. 21. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle.
Transgenic research 2015; 24: 147-153. 2014/09/11. DOI: 10.1007/s11248-014-9832- x. 22. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and
cynomolgus monkeys. Cell Stem Cell 2014; 14: 323-328. 2014/02/18. DOI: 10.1016/j.stem.2014.01.018.
23. Bassett AR, Tibbit C, Ponting CP, et al. Highly efficient targeted mutagenesis of
Drosophila with the CRISPR/Cas9 system. Cell Rep 2013; 4: 220-228. 2013/07/06. DOI: 10.1016/j.celrep.2013.06.020.
24. Friedland AE, Tzur YB, Esvelt KM, et al. Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nature methods 2013; 10: 741-743. 2013/07/03. DOI: 10.1038/nmeth.2532.
25. Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-
Cas system. Nat Biotechnol 2013; 31: 227-229. 2013/01/31. DOI: 10.1038/nbt.2501. 26. Nakayama T, Fish MB, Fisher M, et al. Simple and efficient CRISPR/Cas9-mediated
targeted mutagenesis in Xenopus tropicalis. Genesis 2013; 51: 835-843. 2013/10/15. DOI: 10.1002/dvg.22720.
27. Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of
multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 2013; 31: 684-686. 2013/08/10. DOI: 10.1038/nbt.2652.
28. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9
nucleases. J Mol Cell Biol 2014; 6: 97-99. 2014/01/10. DOI: 10.1093/jmcb/mjt047. 29. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection
of CRISPR/Cas system. Cell research 2014; 24: 372-375. 2014/02/01. DOI: 10.1038/cr.2014.11.
30. Bevacqua RJ, Fernandez-Martin R, Savy V, et al. Efficient edition of the bovine PRNP
prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology 2016; 86: 1886-1896 e1881. 2016/08/28. DOI: 10.1016/j.theriogenology.2016.06.010.
31. Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and
FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 2015; 5: 13878. 2015/09/12. DOI: 10.1038/srep13878.
32. Crispo M, Mulet AP, Tesson L, et al. Efficient Generation of Myostatin Knock-Out
Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PloS one 2015; 10: e0136690. 2015/08/26. DOI: 10.1371/journal.pone.0136690.
33. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via
Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156: 836-843. 2014/02/04. DOI: 10.1016/j.cell.2014.01.027.
34. Zhang Y, Qin W, Lu X, et al. Programmable base editing of zebrafish genome using a
modified CRISPR-Cas9 system. Nat Commun 2017; 8: 118. 2017/07/26. DOI: 10.1038/s41467-017-00175-6.
35. Park DS, Yoon M, Kweon J, et al. Targeted Base Editing via RNA-Guided Cytidine
Deaminases in Xenopus laevis Embryos. Mol Cells 2017; 40: 823-827. 2017/11/28. DOI: 10.14348/molcells.2017.0262.
36. Kim K, Ryu SM, Kim ST, et al. Highly efficient RNA-guided base editing in mouse
embryos. Nat Biotechnol 2017; 35: 435-437. 2017/03/01. DOI: 10.1038/nbt.3816. 37. Ma Y, Yu L, Zhang X, et al. Highly efficient and precise base editing by engineered
dCas9-guide tRNA adenosine deaminase in rats. Cell Discov 2018; 4: 39. 2018/07/25. DOI: 10.1038/s41421-018-0047-9.
38. Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors
in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 2018; 9: 814-819. 2018/08/02. DOI: 10.1007/s13238-018-0568-x. 39. Liu Z, Chen M, Chen S, et al. Highly efficient RNA-guided base editing in rabbit. Nat
Commun 2018; 9: 2717. 2018/07/15. DOI: 10.1038/s41467-018-05232-2. 40. Xie J, Ge W, Li N, et al. Efficient base editing for multiple genes and loci in pigs using
base editors. Nat Commun 2019; 10: 2852. 2019/06/30. DOI: 10.1038/s41467-019- 10421-8.
41. Li G, Zhou S, Li C, et al. Base pair editing in goat: nonsense codon introgression into
FGF5 results in longer hair. The FEBS journal 2019; 286: 4675-4692. 2019/07/06. DOI: 10.1111/febs.14983.
42. Zhou S, Cai B, He C, et al. Programmable Base Editing of the Sheep Genome Revealed
No Genome-Wide Off-Target Mutations. Front Genet 2019; 10: 215. 2019/04/02. DOI: 10.3389/fgene.2019.00215.
43. Zhang W, Aida T, Del Rosario RCH, et al. Multiplex precise base editing in cynomolgus
monkeys. Nat Commun 2020; 11: 2325. 2020/05/13. DOI: 10.1038/s41467-020- 16173-0.
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[D06D0WM 7/84?/0W 8737W H01,6 H0 Supplementary material for
History of genome editing - from meganucleases to CRISPR Simon E. Tröder and Branko Zevnik
Table S1. Supplementary references for in vivo genome editing Method Species Reference
ZFN mouse 1, 2 rat 3 rabbit 4 pig 5, 6 cattle 7, 8 TALEN
worm 9 zebrafish 10, 11
frog 12 rat 13 mouse 14-16 rabbit 17 pig 18, 19
goat 20 sheep 21
cattle 21
non-human primate 22
CRISPR/Cas9
fly 23
worm 24
zebrafish 25
frog 26
rat 27
rabbit 28
pig 29
cattle 30
goat 31
sheep 32
non-human primate 33
CRISPR base editing
zebrafish 34
frog 35
mouse 36
rat 37, 38
rabbit 39
pig 40
goat 41
sheep 42
non-human primate 43
Supplementary references
1. Meyer M, de Angelis MH, Wurst W, et al. Gene targeting by homologous
recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A 2010; 107: 15022-15026. 2010/08/06. DOI: 10.1073/pnas.1009424107. 2. Carbery ID, Ji D, Harrington A, et al. Targeted genome modification in mice using
zinc-finger nucleases. Genetics 2010; 186: 451-459. 2010/07/16. DOI: 10.1534/genetics.110.117002.
3. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of
zinc-finger nucleases. Science 2009; 325: 433. 2009/07/25. DOI: 10.1126/science.1172447.
4. Flisikowska T, Thorey IS, Offner S, et al. Efficient immunoglobulin gene disruption
and targeted replacement in rabbit using zinc finger nucleases. PloS one 2011; 6: e21045. 2011/06/23. DOI: 10.1371/journal.pone.0021045. 5. Hauschild J, Petersen B, Santiago Y, et al. Efficient generation of a biallelic knockout
in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12017. 2011/07/07. DOI: 10.1073/pnas.1106422108.
6. Whyte JJ, Zhao J, Wells KD, et al. Gene targeting with zinc finger nucleases to
produce cloned eGFP knockout pigs. Mol Reprod Dev 2011; 78: 2. 2011/01/27. DOI: 10.1002/mrd.21271.
7. Liu X, Wang Y, Guo W, et al. Zinc-finger nickase-mediated insertion of the lysostaphin
gene into the beta-casein locus in cloned cows. Nat Commun 2013; 4: 2565. 2013/10/15. DOI: 10.1038/ncomms3565.
8. Yu S, Luo J, Song Z, et al. Highly efficient modification of beta-lactoglobulin (BLG)
gene via zinc-finger nucleases in cattle. Cell research 2011; 21: 1638-1640. 2011/09/14. DOI: 10.1038/cr.2011.153.
9. Wood AJ, Lo TW, Zeitler B, et al. Targeted genome editing across species using ZFNs
and TALENs. Science 2011; 333: 307. 2011/06/28. DOI: 10.1126/science.1207773. 10. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using
customized TALENs. Nat Biotechnol 2011; 29: 699-700. 2011/08/09. DOI: 10.1038/nbt.1939.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish
cells using engineered TALENs. Nat Biotechnol 2011; 29: 697-698. 2011/08/09. DOI: 10.1038/nbt.1934.
12. Lei Y, Guo X, Liu Y, et al. Efficient targeted gene disruption in Xenopus embryos using
engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 2012; 109: 17484-17489. 2012/10/10. DOI: 10.1073/pnas.1215421109. 13. Tesson L, Usal C, Menoret S, et al. Knockout rats generated by embryo microinjection
of TALENs. Nat Biotechnol 2011; 29: 695-696. 2011/08/09. DOI: 10.1038/nbt.1940. 14. Panda SK, Wefers B, Ortiz O, et al. Highly efficient targeted mutagenesis in mice
using TALENs. Genetics 2013; 195: 703-713. 2013/08/28. DOI: 10.1534/genetics.113.156570.
15. Sung YH, Baek IJ, Kim DH, et al. Knockout mice created by TALEN-mediated gene
targeting. Nat Biotechnol 2013; 31: 23-24. 2013/01/11. DOI: 10.1038/nbt.2477. 16. Wefers B, Panda SK, Ortiz O, et al. Generation of targeted mouse mutants by embryo
microinjection of TALEN mRNA. Nature protocols 2013; 8: 2355-2379. 2013/11/02. DOI: 10.1038/nprot.2013.142.
17. Song J, Zhong J, Guo X, et al. Generation of RAG 1- and 2-deficient rabbits by embryo
microinjection of TALENs. Cell research 2013; 23: 1059-1062. 2013/07/10. DOI: 10.1038/cr.2013.85.
18. Carlson DF, Tan W, Lillico SG, et al. Efficient TALEN-mediated gene knockout in
livestock. Proc Natl Acad Sci U S A 2012; 109: 17382-17387. 2012/10/03. DOI: 10.1073/pnas.1211446109.
19. Lillico SG, Proudfoot C, Carlson DF, et al. Live pigs produced from genome edited
zygotes. Sci Rep 2013; 3: 2847. 2013/10/11. DOI: 10.1038/srep02847. 20. Cui C, Song Y, Liu J, et al. Gene targeting by TALEN-induced homologous
recombination in goats directs production of beta-lactoglobulin-free, high-human lactoferrin milk. Sci Rep 2015; 5: 10482. 2015/05/23. DOI: 10.1038/srep10482. 21. Proudfoot C, Carlson DF, Huddart R, et al. Genome edited sheep and cattle.
Transgenic research 2015; 24: 147-153. 2014/09/11. DOI: 10.1007/s11248-014-9832- x. 22. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and
cynomolgus monkeys. Cell Stem Cell 2014; 14: 323-328. 2014/02/18. DOI: 10.1016/j.stem.2014.01.018.
23. Bassett AR, Tibbit C, Ponting CP, et al. Highly efficient targeted mutagenesis of
Drosophila with the CRISPR/Cas9 system. Cell Rep 2013; 4: 220-228. 2013/07/06. DOI: 10.1016/j.celrep.2013.06.020.
24. Friedland AE, Tzur YB, Esvelt KM, et al. Heritable genome editing in C. elegans via a
CRISPR-Cas9 system. Nature methods 2013; 10: 741-743. 2013/07/03. DOI: 10.1038/nmeth.2532.
25. Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPR-
Cas system. Nat Biotechnol 2013; 31: 227-229. 2013/01/31. DOI: 10.1038/nbt.2501. 26. Nakayama T, Fish MB, Fisher M, et al. Simple and efficient CRISPR/Cas9-mediated
targeted mutagenesis in Xenopus tropicalis. Genesis 2013; 51: 835-843. 2013/10/15. DOI: 10.1002/dvg.22720.
27. Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of
multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 2013; 31: 684-686. 2013/08/10. DOI: 10.1038/nbt.2652.
28. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9
nucleases. J Mol Cell Biol 2014; 6: 97-99. 2014/01/10. DOI: 10.1093/jmcb/mjt047. 29. Hai T, Teng F, Guo R, et al. One-step generation of knockout pigs by zygote injection
of CRISPR/Cas system. Cell research 2014; 24: 372-375. 2014/02/01. DOI: 10.1038/cr.2014.11.
30. Bevacqua RJ, Fernandez-Martin R, Savy V, et al. Efficient edition of the bovine PRNP
prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system. Theriogenology 2016; 86: 1886-1896 e1881. 2016/08/28. DOI: 10.1016/j.theriogenology.2016.06.010.
31. Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and
FGF5 via zygote injection of CRISPR/Cas9 system. Sci Rep 2015; 5: 13878. 2015/09/12. DOI: 10.1038/srep13878.
32. Crispo M, Mulet AP, Tesson L, et al. Efficient Generation of Myostatin Knock-Out
Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PloS one 2015; 10: e0136690. 2015/08/26. DOI: 10.1371/journal.pone.0136690.
33. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via
Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014; 156: 836-843. 2014/02/04. DOI: 10.1016/j.cell.2014.01.027.
34. Zhang Y, Qin W, Lu X, et al. Programmable base editing of zebrafish genome using a
modified CRISPR-Cas9 system. Nat Commun 2017; 8: 118. 2017/07/26. DOI: 10.1038/s41467-017-00175-6.
35. Park DS, Yoon M, Kweon J, et al. Targeted Base Editing via RNA-Guided Cytidine
Deaminases in Xenopus laevis Embryos. Mol Cells 2017; 40: 823-827. 2017/11/28. DOI: 10.14348/molcells.2017.0262.
36. Kim K, Ryu SM, Kim ST, et al. Highly efficient RNA-guided base editing in mouse
embryos. Nat Biotechnol 2017; 35: 435-437. 2017/03/01. DOI: 10.1038/nbt.3816. 37. Ma Y, Yu L, Zhang X, et al. Highly efficient and precise base editing by engineered
dCas9-guide tRNA adenosine deaminase in rats. Cell Discov 2018; 4: 39. 2018/07/25. DOI: 10.1038/s41421-018-0047-9.
38. Yang L, Zhang X, Wang L, et al. Increasing targeting scope of adenosine base editors
in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 2018; 9: 814-819. 2018/08/02. DOI: 10.1007/s13238-018-0568-x. 39. Liu Z, Chen M, Chen S, et al. Highly efficient RNA-guided base editing in rabbit. Nat
Commun 2018; 9: 2717. 2018/07/15. DOI: 10.1038/s41467-018-05232-2. 40. Xie J, Ge W, Li N, et al. Efficient base editing for multiple genes and loci in pigs using
base editors. Nat Commun 2019; 10: 2852. 2019/06/30. DOI: 10.1038/s41467-019- 10421-8.
41. Li G, Zhou S, Li C, et al. Base pair editing in goat: nonsense codon introgression into
FGF5 results in longer hair. The FEBS journal 2019; 286: 4675-4692. 2019/07/06. DOI: 10.1111/febs.14983.
42. Zhou S, Cai B, He C, et al. Programmable Base Editing of the Sheep Genome Revealed
No Genome-Wide Off-Target Mutations. Front Genet 2019; 10: 215. 2019/04/02. DOI: 10.3389/fgene.2019.00215.
43. Zhang W, Aida T, Del Rosario RCH, et al. Multiplex precise base editing in cynomolgus
monkeys. Nat Commun 2020; 11: 2325. 2020/05/13. DOI: 10.1038/s41467-020- 16173-0.
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