Site-specific mutation

• Target mutagenesis is to make DNA sequence changes for a target gene • Two methods of site-specific mutation - Homologous recombination (HR) - Engineered enzymes for gene editing ü Zinc-finger nucleases (ZFNs) ü Transcription activator-like effector nucleases (TALENs) ü CRISPR-Cas Homologous recombination

• A type of genetic recombination in which nucleotide sequences are exchanged between two similar DNA fragments or homologous chromosomes • Homologous recombination produces new combinations of DNA sequences during meiosis • Homologous recombination is used by cells to accurately repair DNA double-strand breaks Meiosis and homologous recombination Homologous Recombination (HR) induce targeted gene mutation in yeast Transformation in yeast

• Electroporation: electric pulse results in the formation of transient pores in the cell membrane, allowing the entry of macromolecules • https://dnalc.cshl.edu/resources/animations/ Yeast deletion project

• Yeast (single cell organism), first genome sequence of an eukaryote, has genome size of 12 Mb and 5,885 protein-coding genes • Saccharomyces Genome Deletion Project using homologous recombination (HR) created a complete, systematic deletion collections to identify essential genes and understand gene function – http://www-sequence.stanford.edu/group/yeast_deletion_project/ deletions3.html • More than 21,000 mutant strains that carry precise deletions of all genes

Giaever et al. Nature 2002; Genetics 2014 Phenotypic annotations of 3,489 genes

Giaever et al., Genetics 2014 A global genetic interaction network maps a wiring diagram of cellular function in yeast

• Genetic interaction: mutations in two genes produce a phenotype that is surprising in light of each mutation's individual effects. This phenomenon, which defines genetic interaction, can reveal functional relationships between genes and pathways • An extreme negative or lethal genetic interaction occurs when two mutations, neither lethal individually, combine to cause cell death Genetic interaction

No interacon Interacon

Fitness AB (100%) AC (100%) 100% 100% A- (80%) A- (80%) -B (70%) -C (70%)

50% 50% -- (50%)

-- (20%) A global genetic interaction network maps a wiring diagram of cellular function in yeast

• Crossed single-gene mutants and produced ~23 millions of double gene mutants • Identified 1 million genetic interactions • Only ~1000 genes in yeast are individually essential in standard growth conditions and cause lethality when mutated • ~10,000 lethal interactions between nonessential gene pairs were found

Costanzo et al., Science 2016 Homologous recombination (HR) does not work well in plants

• High frequency of DNA integration at non-homologous sites by illegitimate recombination • Typically 105 to 107 illegitimate recombination events for every homologous recombination event • It is impractical to screen large numbers of transformed plants to identify the rare individuals that have undergone homologous recombination

Wright et al., The Plant Journal 2005 Homologous recombinaon

Illegimate or off-target recombinaon Gene or genome editing

• About 20 years ago, gene or genome editing came to a new era with the discovery that nucleases could be engineered to create site-specific DNA double-strand breaks that could stimulate homologous recombination more than 1000-fold

Bak et al., Trends in genetics 2018 Gene or genome editing

• Double-strand breaks can be repaired through homologous recombination (HR) or non-homologous end joining (NHEJ) • Precise gene repair by HR using an exogenously supplied template with homology to the targeted site • Error-prone non-homologous end joining (NHEJ) creates INDELs

Bak et al., Trends in genetics 2018 DNA double strand break and repair

Non-homologous Homologous end joining recombinaon repair

Urnov et al. Nature review genetics 2010 Gene editing

• Key requirements for the engineered nucleases – Precise recognition of target sequence and low level of off-target site mutations – Easy to be engineered for user-defined sequences with low cost so that any gene can be edited – Practicable for any species Zinc-Finger domain

• Zinc-Finger domain, a DNA binding domain of a transcription factor • Tandem repeats of such zinc fingers can be used to target desired genomic DNA sequences Zinc-Finger Nucleases (ZFNs)

• Engineered Zinc-finger domains fused to a DNA cleavage domain of a restriction enzyme • One finger recognize 3bp DNA sequence, up to 6 fingers each side • Two adjacent binding events occur in the correct orientation and with appropriate spacing for dimer formation Ø One Zinc-Finger recognizes 3 bp, not flexible or easy to be designed for broad target sequences

Urnov et al. Nature review genetics 2010 Transcription Activator-Like Effectors (TALE)

• TALEs, a transcriptional activator produced by a plant pathogen • Regulate plant genes and increases plant susceptibility to pathogen, or trigger plant defense

Bogdanove et al., Current Opinion in Plant Biology 2010 Transcription Activator-Like Effectors (TALE)

• DNA binding domain contains 30 tandem repeats of a 33- to 35-amino-acid- sequence motif • The amino acid sequence of each repeat is invariant, with the exception of two adjacent amino acids (the repeat variable diresidue, RVD) • RVD recognize different DNA base, one-to-one match between the RVDs and the nucleotides

Christian et al., Genetics 2010 Transcription Activator-Like Effectors Nucleases (TALEN)

• Modularity of the repeats enables rapid construction of TALE nucleases (TALENs) with novel specificities to target double-strand breaks at specific locations

Christian et al., Genetics 2010 Example: Improving cold storage and processing traits in potato through gene editing

• Cold storage of potato is commonly used to reduce sprouting and extend postharvest shelf life, but accumulate reducing sugars (e.g., glucose and fructose)

• The reducing sugars interacted with free amino acid cause brown, bitter-tasting products and elevated levels of acrylamide (a potential carcinogen) Example: Improving cold storage and processing traits in potato through TALEN editing

• Vacuolar invertase gene (Vlnv) encodes a protein that breaks down sucrose to glucose and fructose Improving cold storage and processing traits in potato through targeted gene knockout

• TALENs was used to knockout VInv within the commercial potato variety, Ranger Russet

Clasen et al., Plant Biotechnology 2015 Improving cold storage and processing traits in potato through targeted gene knockout

• Protoplasts from leaves on 3-week- old potato plants were isolated and transformed with plasmids encoding TALEN pairs

Clasen et al., Plant Biotechnology 2015 Improving cold storage and processing traits in potato through targeted gene knockout

Clasen et al., Plant Biotechnology 2015 Example: Improving cold storage and processing traits in potato through TALEN editing

Clasen et al., Plant Biotechnology 2015 Discovery of CRISPR/Cas9 in immunity system

Horvath and Barrangou, Science 2010 CRISPR/Cas9 can be programed for a target DNA sequence

• The Cas9 is guided by two-RNA structure, tracrRNA and targeting crRNA • The target sequence consists of a 20-bp DNA sequence complementary to the gRNA, followed by trinucleotide sequence (5'-NGG-3') called the protospacer adjacent motif (PAM) • The Cas nuclease digests both strands of the genomic DNA 3-4 nucleotides 5' of the PAM sequence

Jinek et al., Science 2012 Example: High-throughput functional genetics using CRISPR/Cas9

• Melanoma is the most dangerous type of skin cancer. Globally, in 2012, it occurred in 232,000 people and resulted in 55,000 deaths • ~60% of melanomas carry an activating mutation in the gene encoding the serine–threonine protein kinase B- RAF (BRAF) • 90% of reported BRAF mutations result in a substitution of glutamic acid for valine at amino acid 600 (the V600E mutation)

Flaherty et al., 2010 Example: High-throughput functional genetics using CRISPR/Cas9

• Vemurafenib (known as PLX4032) – A small molecule, is an inhibitor of BRAF with the V600E mutation – Causes programmed cell death in melanoma cell – Vemurafenib received FDA approval for the treatment of late-stage melanoma on August 17, 2011

• Most side effects appeared to be proportional to the dose

Flaherty et al., 2010 Example: High-throughput functional genetics using CRISPR/Cas9

• What genes are related to the medicine (PLX) responses? • A genome-scale CRISPR/Cas9 knockout (GeCKO) library targeting 18,080 genes, 5 targets per gene • Transduced the human melanoma cell line A375 • Screen for genes whose loss is involved in resistance to vemurafenib

Shalem et al., 2014 Science Example: High-throughput functional genetics using CRISPR-cas9

• Loss of function of NF2 cause resistance to PLX – All 5 targets of the gene showed resistance, not false positive error

Shalem et al., 2014 Science Gene-edited mushroom escapes US regulation

• “The mushroom can be cultivated and sold without passing through the agency’s regulatory process — making it the first CRISPR-edited organism to receive a green light from the US government.”

– Waltz, E., 2016. Gene-edited CRISPR mushroom escapes US regulation. Nature News, April 21 Gene editing is different from GMO

GMO Gene eding

Target gene CRISPR-Cas9

Substuon, Transformaon small inseron, Transformaon or deleon

Selfing or Selfing or backcross backcross References • Bak, R.O., Gomez-Ospina, N. and Porteus, M.H., 2018. Gene editing on center stage. Trends in Genetics, 34(8):600-611. • Bogdanove, A.J., Schornack, S. and Lahaye, T., 2010. TAL effectors: finding plant genes for disease and defense. Current opinion in plant biology, 13(4): 394-401. • Christian, Michelle, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186.2 (2010): 757-761. • Clasen, B.M., Stoddard, T.J., Luo, S., Demorest, Z.L., Li, J., Cedrone, F., Tibebu, R., Davison, S., Ray, E.E., Daulhac, A. and Coffman, A., 2016. Improving cold storage and processing traits in potato through targeted gene knockout. Plant biotechnology journal, 14(1):169-176. • Costanzo, M., VanderSluis, B., Koch, E.N., Baryshnikova, A., Pons, C., Tan, G., Wang, W., Usaj, M., Hanchard, J., Lee, S.D. and Pelechano, V., 2016. A global genetic interaction network maps a wiring diagram of cellular function. Science, 353(6306). References

• Flaherty, K.T., Puzanov, I., Kim, K.B., Ribas, A., McArthur, G.A., Sosman, J.A., O'Dwyer, P.J., Lee, R.J., Grippo, J.F., Nolop, K. and Chapman, P.B., 2010. Inhibition of mutated, activated BRAF in metastatic melanoma. New England Journal of Medicine, 363(9), pp.809-819. • Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, S., Lucau-Danila, A., Anderson, K., Andre, B. and Arkin, A.P., 2002. Functional profiling of the Saccharomyces cerevisiae genome. nature, 418(6896):387. • Giaever, G. and Nislow, C., 2014. The yeast deletion collection: a decade of functional genomics. Genetics, 197(2):451-465. • Horvath, Philippe, and Rodolphe Barrangou. CRISPR/Cas, the immune system of bacteria and archaea. Science 327.5962 (2010):167-170. • Jinek, Martin, et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337.6096 (2012):816-821. References

• Jinek, Martin, et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337.6096 (2012):816-821. • Shalem, O., Sanjana, N.E., Hartenian, E., Shi, X., Scott, D.A., Mikkelsen, T.S., Heckl, D., Ebert, B.L., Root, D.E., Doench, J.G. and Zhang, F., 2014. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science, 343(6166):84-87. • Shalem, O., Sanjana, N.E. and Zhang, F., 2015. High-throughput functional genomics using CRISPR–Cas9. Nature Reviews Genetics, 16(5):299. • Urnov, Fyodor D., et al. Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics 11.9 (2010):636-646. • Waltz, E., 2016. Gene-edited CRISPR mushroom escapes US regulation. Nature News, 532:293. • Wright, D.A., Townsend, J.A., Winfrey Jr, R.J., Irwin, P.A., Rajagopal, J., Lonosky, P.M., Hall, B.D., Jondle, M.D. and Voytas, D.F., 2005. High‐ frequency homologous recombination in plants mediated by zinc‐finger nucleases. The Plant Journal, 44(4), pp.693-705. •