Targeted Gene Modification Genomics 42925 Hariklia Karageorgiou Targeted

Targeted Gene Modification Genomics (42925) Hariklia Karageorgiou

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

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

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

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.

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.

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 treat human diseases. BMC medicine, 8(1), 42.

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.

Molecular analysis of heat shock-induced transgenics PCR products -/+ Eco. RI 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.

Targeted mutations at maize IPK 1 • IPK 1 is an important target gene for modification (phytate is an antinutrient) • Can either KO IPK 1 activity alone, or simultaneously add a new gene (‘trait stacking’) • IPK 1 and IPK 2 are 98% identical • ZFN targeted paralog-specific sequences • Specificity tested in yeast • Targeted exon 2 sequences with ZFN 12 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.

NHEJ at IPK 1 induced by ZFNs • Initially tested capacity of ZFNs to induce mutations through NHEJ at IPK 1 • NHEJ induces deletions or insertions at the target site that result in loss of function • Deep sequencing of IPK 1 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.

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 (Avr. Bs 3 and Pth. Xo 1). • 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 Fok. I 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.

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, gkr 218.

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.

CRISPR-mediated gene modification in humans • Normally the Cas 9 endonuclease forms a complex with two short RNA molecules, CRISPR RNA (cr. RNA) and transactivating cr. RNA (tracr. RNA) 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 sg. RNA, composed of functional portions of cr. RNA and tracr. RNA, to form a targeted RNA-guided endonuclease (RGEN). • Human CCR 5 encodes an essential co-receptor of HIV and is a potential target for the treatment for AIDS. • The Cas 9 target sequence consists of a 20 -bp DNA sequence complementary to the cr. RNA or the chimeric RNA and the trinucleotide (5′-NGG-3′) protospacer adjacent motif (PAM) recognized by Cas 9. • The targeting complex consists of the Cas 9 nuclease plus a chimeric ss. RNA molecule that encodes cr. RNA (RED) and a 20 base sequence that specifies the target sequence in the CCR 5 gene (Blue) • The DSB induced by Cas 9 is shown by the RED arrowheads Cho et al. , (2013). Targeted genome engineering in human cells with the Cas 9 RNA-guided endonuclease. Nature biotechnology, 31(3), 230 -232.

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

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 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. Cermak, T. , Doyle, E. L. , Christian, M. , Wang, L. , Zhang, Y. , Schmidt, C. , & Voytas, D. F. (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic acids research, gkr 218. Cho, S. W. , Kim, S. , Kim, J. M. , & Kim, J. S. (2013). Targeted genome engineering in human cells with the Cas 9 RNA-guided endonuclease. Nature biotechnology, 31(3), 230 -232. 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. Davis, D. , & Stokoe, D. (2010). Zinc finger nucleases as tools to understand treat human diseases. BMC medicine, 8(1), 42. 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. Kim, S. , & Kim, J. S. (2011). Targeted genome engineering via zinc finger nucleases. Plant biotechnology reports, 5(1), 9 -17. 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. 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 Moscou, M. J. , & Bogdanove, A. J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science, 326(5959), 1501 -1501. Mussolino, C. , & Cathomen, T. (2013). RNA guides genome engineering. Nature biotechnology, 31(3), 208 -209. 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. Shukla, V. K. , Doyon, Y. , Miller, J. C. , De. Kelver, 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. 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. Zhang et al. , (2011). Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nature biotechnology, 29(2), 149 -153.