1. Targeted Gene Modification Genomics (42925) Hariklia Karageorgiou

2. Targeted mutagenesis using embryonic stem (ES) cells Used to inactivate genes to investigate function (X - ) Lodish et al., Molecular cell biology. (2004) Molecular genetic techniques and genomics. Chap. 9 p. 351-403. New York: W.H. Freeman and Company

3. If transgenic ES cells contribute to germ line, crossing chimeras to wt mice will result in heterozygous off-spring. ES cells heterozygous for a disrupted gene are used to produce homozygous ‘knock outs’ Genetic chimeras are easily identified according to coat colour Lodish et al., Molecular cell biology. (2004) Molecular genetic techniques and genomics. Chap. 9 p. 351-403. New York: W.H. Freeman and Company

4. ES cells heterozygous for a disrupted gene are used to produce homozygous ‘knock outs’ Investigate phenotype (Approx 25%) Only 50% of brown progeny will contain the transgene Molecular screening to identify X - /X + heterozygotes Lodish et al., Molecular cell biology. (2004) Molecular genetic techniques and genomics. Chap. 9 p. 351-403. New York: W.H. Freeman and Company

5. Targeted genome modification in mammalian cells Capecchi, M. R. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nature Reviews Genetics, 6(6), 507-512.

6. Technologies for achieving targeted gene modification Zinc Finger Nucleases (ZFNs) Transcription Activator-Like Effector Nucleases (TALENs) Type II clustered, regularly interspaced, short palindromic repeat system (CRISPR) (provides prokaryotes with adaptive immunity to viruses and plasmids) Mussolino, C., & Cathomen, T. (2013). RNA guides genome engineering. Nature biotechnology, 31(3), 208-209.

7. Site-specific modifications with meganucleases Nucleases induce site-specific double-strand breaks triggering: Mutagenic NHEJ Small changes in target gene sequence (HR) Gene replacement (HR) Davis, D., & Stokoe, D. (2010). Zinc finger nucleases as tools to understand and treat human diseases. BMC medicine, 8(1), 42.

8. Targeted mutagenesis using ZFNs QQR – gene encoding ZFN QBS – ZFN target sequences Lloyd, A., Plaisier, C. L., Carroll, D., & Drews, G. N. (2005). Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 102(6), 2232-2237.

9. Molecular analysis of heat shock-induced transgenics PCR products -/+ EcoRI treatment High frequency of target site modification by NHEJ Includes insertions and deletions around ZFN target sequence Highly efficient targeted mutagenesis Lloyd, A., Plaisier, C. L., Carroll, D., & Drews, G. N. (2005). Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 102(6), 2232-2237.

10. Targeted mutations at maize IPK1 IPK1 is an important target gene for modification (phytate is an antinutrient) Can either KO IPK1 activity alone, or simultaneously add a new gene (‘trait stacking’) IPK1 and IPK2 are 98% identical ZFN targeted paralog-specific sequences Specificity tested in yeast Targeted exon 2 sequences with ZFN12 Shukla et al., (2009). Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, 459(7245), 437-441. Kim, S., & Kim, J. S. (2011). Targeted genome engineering via zinc finger nucleases. Plant biotechnology reports, 5(1), 9-17.

11. NHEJ at IPK1 induced by ZFNs Initially tested capacity of ZFNs to induce mutations through NHEJ at IPK1 NHEJ induces deletions or insertions at the target site that result in loss of function Deep sequencing of IPK1 PCR products from ZFN transformed cells Urnov et al., (2010). Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 11(9), 636-646. Shukla et al., (2009). Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, 459(7245), 437-441.

12. TALENs TALEs – Transcription Activator-Like Effector proteins produced by certain plant pathogenic bacteria. Act as effectors during pathogenesis. Secreted through the TTSS pathway and target host nuclear gene expression (AvrBs3 and PthXo1). Contain a NLS, N-terminal translocation signal and transcriptional activator domain Contain 30 tandem repeats of a 33-35 aa motif that can recognise a single base through two di-variant residues (RVD) which has been deciphered Can be fused to the FokI nuclease for targeted DSBs Moscou, M. J., & Bogdanove, A. J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science, 326(5959), 1501-1501.

13. TALE engineering TALE genes can be mutated to generate sequence-specific DNA binding proteins The modified TALEs can be fused to nucleases for targeted DSBs in plants and animals Modified TALEs can be fused to transcriptional activators to trigger ectopic gene expression in mammals Zhang et al., (2011). Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nature biotechnology, 29(2), 149-153. Christian et al., (2010). Targeting DNA double-strand breaks with TAL effector nucleases. Genetics, 186(2), 757-761. Cermak et al., (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic acids research, gkr218.

14. Cracking the TALE code Moscou, M. J., & Bogdanove, A. J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science, 326(5959), 1501-1501.

15. CRISPR-mediated gene modification in humans Normally the Cas9 endonuclease forms a complex with two short RNA molecules, CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) which guide the enzyme to recognize and cleave a site in non-self DNA (e.g. in a bacteriophage genome). The two short RNAs can be replaced by a chimeric sgRNA, composed of functional portions of crRNA and tracrRNA, to form a targeted RNA-guided endonuclease (RGEN). Human CCR5 encodes an essential co-receptor of HIV and is a potential target for the treatment for AIDS. The Cas9 target sequence consists of a 20-bp DNA sequence complementary to the crRNA or the chimeric RNA and the trinucleotide (5′-NGG-3′) protospacer adjacent motif (PAM) recognized by Cas9. The targeting complex consists of the Cas9 nuclease plus a chimeric ssRNA molecule that encodes crRNA (RED) and a 20 base sequence that specifies the target sequence in the CCR5 gene (Blue) The DSB induced by Cas9 is shown by the RED arrowheads Cho et al., (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nature biotechnology, 31(3), 230-232.

16. Summary Traditional gene modification approaches have been dependent upon mutagenesis, or transformation processes Targeted gene modification can be achieved in mouse through transformation of ES cells Targeted gene replacement in plants was not possible until the advent of ZFNs Precise gene replacement/modification will have significant applications in agriculture Similar technologies are being used to develop somatic gene therapies in animal cells

17. References 1.Capecchi, M. R. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nature Reviews Genetics, 6(6), 507-512. 2.Cermak, T., Doyle, E. L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., & Voytas, D. F. (2011). (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic acids research, gkr218. 3.Cho, S. W., Kim, S., Kim, J. M., & Kim, J. S. (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nature biotechnology, 31(3), 230-232. 4.Christian M., Cermak, T., Doyle, E. L., Schmidt, C., Zhang, F., Hummel, A., & Voytas, D. F. (2010). Targeting DNA double-strand breaks with TAL effector nucleases. Genetics, 186(2), 757-761. 5.Davis, D., & Stokoe, D. (2010). Zinc finger nucleases as tools to understand and treat human diseases. BMC medicine, 8(1), 42. 6.Gaj, T., Gersbach, C. A., & Barbas III, C. F. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in biotechnology, 31(7), 397-405. 7.Kim, S., & Kim, J. S. (2011). Targeted genome engineering via zinc finger nucleases. Plant biotechnology reports, 5(1), 9-17. 8.Lloyd, A., Plaisier, C. L., Carroll, D., & Drews, G. N. (2005). Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 102(6), 2232-2237. 9.Lodish H, Berk A, Matsudaira P. Molecular cell biology. (2004) Molecular genetic techniques and genomics. Chap. 9 p. 351-403. New York: W.H. Freeman and Company 10.Moscou, M. J., & Bogdanove, A. J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science, 326(5959), 1501-1501. 11.Mussolino, C., & Cathomen, T. (2013). RNA guides genome engineering. Nature biotechnology, 31(3), 208-209. 12.Radecke, F., Peter, I., Radecke, S., Gellhaus, K., Schwarz, K., & Cathomen, T. (2004). Targeted chromosomal gene modification in human cells by single-stranded oligodeoxynucleotides in the presence of a DNA double-strand break. Molecular Therapy, 14(6), 798-808. 13.Shukla, V. K., Doyon, Y., Miller, J. C., DeKelver, R. C., Moehle, E. A., Worden, S. E.,... & Urnov, F. D. (2009). Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature, 459(7245), 437-441. 14.Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., & Gregory, P. D. (2010). Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 11(9), 636-646. 15.Zhang et al., (2011). Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nature biotechnology, 29(2), 149-153.