Genome Engineering

1. Genome Engineering

2. Why engineer the genome? Eliminate gene activity -test loss of function phenotype -spatial control of gene loss(mosaic analysis) -temporal control of gene loss Control gene expression -investigation of gene function -structure/function analysis of gene activity -labeling specific cell types (lineage analysis/fate mapping, in vivo imaging) -killing specific cell types Test the function of genome organization elements -enhancers, insulators, etc.

3. “Genetic Engineering” Revolution Goal: Manipulate pieces of DNA to build recombinant constructs with a defined composition and sequence Approach: “Steal” enzymes, vectors and other tricks from “simpler” systems Tools -Enzymes: restriction enzymes, DNA ligases, RNA and DNA polymerases, etc. -Vectors: Plasmids, bacteriophage (lambda, P1), BACs, YACs -Hosts: bacteriophage and bacteria (sometimes yeast) In order to create: -cDNA and genomic libraries to isolate DNA of interest -Recombinant plasmids for manipulating genes, protein expression, site-directed mutagenesis, etc.

4. Genome Engineering Revolution Goals: Manipulate the genome of “higher” organisms, including humans, to facilitate more sophisticated genetics and therapies. Approach: Use Genetic Engineering to build DNA of interest and use new tricks for introducing and manipulating foreign DNA in the genome. Requirements: -Genetic system -Ways to get engineered DNA back into chromosomes in germline Tools -Enzymes: integrase/recombinases, transposases, transcription factors -Vectors: transposons, viruses -Cellular pathways: DNA repair/homologous recombination -Hosts: Genetically tractable model organisms, humans In order to: -Identify new genes -Mutate genes of interest -Study regulatory DNA and gene expression -Ectopically express foreign or modified genes

5. Exploiting “Normal” Cellular Processes Transcription -in vitro: RNA polymerases -in vivo: controlled expression of genes of interest Replication -in vitro: DNA polymerases, ligases, site-directed mutagenesis, PCR -in vivo: recombinant DNA--plasmids, BACs, YACs Phages, viruses, transposons -in vitro: simplest enzymes, reverse transcriptase, “Gateway” cloning -in vivo: insertion of foreign DNA into genomes, mobilizing DNA elements Host Genome Defense -in vitro: Restriction enzymes -in vivo: siRNA response DNA Repair -in vitro: endonucleases, etc. -in vivo: recombineering, homologous “recombination”

6. Getting DNA Into a Cell (That Can Form a Germ Cell) 1) Injection -inject into syncytial gonad in C. elegans -inject into the syncytial embryo in Drosophila -inject into the nucleus of mouse oocyte 2) Infection Use viruses to infect cells or embryos -can be used for transient gene delivery -can “go germline” if use ES cells or infect right cells of embryo -used for insertional mutagenesis in mouse and fish 3) Transfection Cells will take up DNA under correct treatment e.g. -when DNA is in calcium phosphate ppt. -when DNA is incorporated in liposomes (lipofection) -when cells are exposed to electric field (electroporation)

7. Getting DNA Into a Genome 1) Random Integration -Linearized DNA will often incorporate randomly into the genome -Often incorporates in multiple copies -Can be enhanced by using Restriction Enzyme Mediated Integration (REMI) Supply cells with same RE used to linearize DNA 2) Transposon or Retroviral Insertion -Place DNA of interest in transposon or retroviral “backbone” -Supply DNA + Transposase OR Produce packaged virus and infect cells -Usually get single integration -Integration may not be random, depending on insertion preferences of system

8. Transposons -Mobile genetic elements that can “jump” around genome -DNA transposons utilize “Cut and paste” mechanism for jumping (remain relatively low copy #) -Retrotransposons utilize RNA intermediate and “copy and paste” tranposition (increases copy # --LINE transposon alone makes up 21% of human genome!) -Transposons can be separated from transposase so transposition is controlled independently -Transposons can be vehicles for transgene delivery and mobilization

9. Requirements: -DNA of interest cloned into vector between transposon inverted repeats -Dominant selectable marker to screen for transformants -Separate source of transposase--can control when transposons jump Inject vector + transposase and construct will “hop” off plasmid and into genome

10. Transposons as Mutagens Advantage: mutated gene is “tagged” by transposon for easy identification Disadvantage: transposons do not insert randomly -some genes hit easily, others not

11. Commonly Used Transposons Species of StudyTransposonOriginal source Drosophila P element D. melanogaster piggy bac* Insects (cabbage looper) minos* D. hydei *works in many species--including mouse C. Elegans Tc1 C. elegans note: used for mutagenesis but not transgenesis Zebrafish Tol2 Fish (medaka) Mouse Sleeping Beauty Fish (salmon/trout)

12. Gal4 lacZ Identify interesting genes by expression pattern Modified Screening Vectors: The Enhancer Trap

13. Viruses as Vectors for Genome Engineering Advantages: -Can act as vehicle to deliver DNA into cell AND into genome -Can be modified in similar ways as transposons Also useful for transient delivery of DNA into cells -Express gene/construct of interest during development by exposing embryo to virus -Gene therapy by expressing gene/construct of interest transiently in human cells Disadvantages: Can’t always supply virus to germ cells More difficult to control than transposons (can’t jump them around)

14. Getting DNA Into a Genome 1) Random Integration -Linearized DNA will often incorporate randomly into the genome -Often incorporates in multiple copies -Can be enhanced by using Restriction Enzyme Mediated Integration (REMI) Supply cells with same RE used to linearize DNA 2) Transposon or Retroviral Insertion -Place DNA of interest in transposon or retroviral “backbone” -Supply DNA + Transposase OR Produce packaged virus and infect cells -Usually get single integration -Integration may not be random, depending on insertion preferences of system 3) Integration into a Defined “Landing Site” -Combine DNA of interest with integration sequence (e.g. from bacteriophage) -Use host whose genome has already been modified to contain landing site -Supply DNA + Integrase

15. -Normally used by bacteriophage for lysogenic cycle -Promote SITE-SPECIFIC integration -Many systems have: -Single Integrase with no additional factors needed -Works in vitro and in other hosts (flies, mice) -Some are irreversible (and therefore VERY efficient) “forward” rxn requires only integrase “reverse” rxn requires integrase + excisionase -Basis for “Gateway” cloning system -CRE/lox system is another example from bacteriophage P1 -FLP/FRT system is related system from Yeast

16. Integrases/Recombinases Advantages: -Specific insertion site -Very efficient (with irreversible integrases) -easier transformation and larger constructs Disadvantages: -Need target sequence in genome -Specific insertion site -can’t do random integrations/screens -Can’t mobilize after integration

17. Getting DNA Into a Genome 1) Random Integration -Linearized DNA will often incorporate randomly into the genome -Often incorporates in multiple copies -Can be enhanced by using Restriction Enzyme Mediated Integration (REMI) Supply cells with same RE used to linearize DNA 2) Transposon or Retroviral Insertion -Place DNA of interest in transposon or retroviral “backbone” -Supply DNA + Transposase OR Produce packaged virus and infect cells -Usually get single integration -Integration may not be random, depending on insertion preferences of system 3) Integration into a Defined “Landing Site” -Combine DNA of interest with integration sequence (e.g. from bacteriophage) -Use host whose genome has already been modified to contain landing site -Supply DNA + Integrase 4) Homologous Recombination/ Gene Targeting -Homologous regions of DNA can be targeted for recombination -Allows insertion of DNA construct in defined site -Used to “knock out” or modify gene of interest -Low frequency event: need to be able to screen large numbers of events (e.g. in ES cells in culture)

18. Curent Methods of Choice C. Elegans -inject DNA into syncytial gonad -get multicopy extrachromosomal arrays of DNA Drosophila -inject DNA into posterior pole of embryo (germ plasm) -use transposon backbone + transposase OR -phage integration site + integrase -homologous recombination Zebrafish -Tol2 transposon backbone + transposase Mouse -inject DNA into oocyte nucleus for random integration -transfect ES cells and screen for homologous recombination when need targeted insertion

19. Directed Modification of the Genome Modifications to endogenous loci

20. Zn Finger Nucleases

21. Gene Targeting by Homologous Recombination “Knock out” “Knock in” GFP Gene X GFP http://nobelprize.org/

22. Directed Modification of the Genome Modifications to endogenous loci -Possible in mouse and Drosophila Modifications to “exongenous” loci -i.e. locus reconstruction and modification

23. Modifying region of interest w/o restriction enzymes Recombineering: new methods for genetic engineering Limitation to traditional “cloning”: Number of useful restriction enzyme sites that are present or can be introduced is limited, especially with large constructs Solution: Cloning without restriction enzymes by RECOMBINEERING “Sub-cloning” large regionof interest w/o restriction enzymes Region of interest can be 100kb or more 50 bp homology arms http://recombineering.ncifcrf.gov/

24. Tools for Genome Engineering -Recombinases

25. Site-specific Recombinases: Precision tools for genome modification FLP Recombinase and FLP Recombinase Targets (FRT) in Drosophila Stolen from yeast by Golic and Lindquist, 1989 Cre Recombinase and loxP target sites in Mouse Stolen from bacterophage P1 by Sauer and Henderson, 1988 Garcia-Otin and Guillou, 2006

26. Using Recombinase Sites in cis to Excise DNA 1) Turn on a gene of interest only in cells that express the recombinase Promoter YFG STOP Promoter YFG

27. FLiPing Out YFG (Your Favorite Gene) TXN Stop Promoter can be general or tissue-specific FRT sites (or loxP sites) (or CRE) Recombinase can be tissue-specific Recombinase can be conditional (e.g. hormone activated) Struhl and Basler, 1993 YFG is only expressed after recombinase excises transcription STOP Examples Lineage analysis--mark all cells ever expressing recombinase YFG is lacZ or GFP, promoter is general (on in most/all cells) Recombinase can be tissue-specific Precise control of YFG gene expression -YFG is only expressed in which promoter is active and recombinase has also been expressed

28. Using Recombinase Sites in cis to Excise DNA 1) Turn on a gene of interest only in cells that express the recombinase Promoter YFG STOP Promoter YFG 2) Delete your favorite gene (YFG) only in cells expressing recombinase YFG

29. Tissue Specific Gene Inactivation -Create allele of gene flanked by loxP sites (“floxed”) (need one large or two smaller targeting constructs) -Verify that floxed allele still has wt activity -Generate the following genotype: Gene Xfloxed + Cell-type specific Cre expression Gene X∆ -Region of gene b/w loxP sites is excised only where Cre is expressed “Floxed” allele Deletion allele

30. Using Recombinase Sites in cis to Excise DNA 1) Turn on a gene of interest only in cells that express the recombinase Promoter YFG STOP Promoter YFG 2) Delete your favorite gene (YFG) only in cells expressing recombinase YFG 3) Recombination Mediated Cassette Exchange Endogenous genomic region on chromosome YFG YFG* Engineered genomic region (on BAC) Modified endogenous genomic region on chromosome YFG*

31. Using Recombinase Sites in trans to Promote Recombination b/w Chromosomes 1) Mitotic recombination to make clones of homozygous mutant cells in a heterozygous background

32. Mitotic Recombination Genetic mosaics can be created by mitotic recombination induced by X-rays or a site-specific recombinase FLP/FRT in Drosophila: Golic and Lindquist, 1989 Essential reagents: Xu and Rubin, Chou and Perrimon Homozygous Mutant Daughter Cell Heterozygous Mother Cell J. Treisman

33. Using Recombinase Sites in trans to Promote Recombination b/w Chromosomes 1) Mitotic recombination to make clones of homozygous mutant cells in a heterozygous background 2) Lineage tracing by “repairing” a split marker gene tubulin promoter lacZ tubulin promoter lacZ

34. Using Recombinase Sites in trans to Promote Recombination b/w Chromosomes 1) Mitotic recombination to make clones of homozygous mutant cells in a heterozygous background 2) Lineage tracing by “repairing” a split marker gene tubulin promoter lacZ tubulin promoter lacZ 3) “Designer” Duplications and Deletions c c a a b c a b c a b b

35. Tools for Genome Engineering -Recombinases -Exongenous transcription factors -activators -repressors -responsive promoters

36. Controlling Gene Expression Reasons to drive expression of a gene of interest: -Label a cell type for identification, purification or lineage analysis -Kill or alter the function of a cell type of interest (e.g. express toxin in specific cell) -Observe an over-expression or dominant negative phenotype -Knock down your gene’s function (e.g. RNAi) -Rescue a mutant phenotype (e.g. to identify gene responsible) -Determine in which cells your gene is required to function -Conduct a structure-function analysis of your protein -Perform gene therapy

37. “Binary” expression systems Tissue-specific Promoter “Binary system” allows same set of Gal4 “driver” stocks to be used to express any gene of interest Related systems: -lexA -Tet -qUAS

38. “Intersectional” systems for increased spatial control Combining activators and repressors: genes are expressed where activators present AND repressors are absent Split activators: genes are expressed only where Activation domain AND DNA binding domain are expressed together

39. Gal4/UAS With Temporal Control Temperature sensitive Gal80 repressor Hormone-responsive Gal4

40. Tools for spatial and temporal control of gene expression: 1) Utilize exogenous transcription factors and recombinases to control gene expression -Gal4/UAS -LexA -QF-qUAS -TET On/Off -FLP/FRT -CRE/LOX 2) Use specific PROMOTERS to control spatial expression of transcription factors and recombinases -Engineered promoter fragments -Random “enhancer traps” -Knock in to recapitualate endogenous pattern 3) Use inducible transcription factors/recombinases for TEMPORAL control -Switch-Gal4 (hormone responsive activator) -Temperature-sensitive Gal80 (repressor) -QF—(quinic acid responsive activator) -TET (Tetracycline responsive) -CRE-ER (hormone responsive CRE)

41. FLiPing Out YFG (Your Favorite Gene) TXN Stop Promoter can be general or tissue-specific FRT sites (or loxP sites) (or CRE) Recombinase can be tissue-specific Recombinase can be conditional (e.g. hormone activated) Struhl and Basler, 1993 YFG is only expressed after recombinase excises transcription STOP Examples Lineage analysis--mark all cells ever expressing recombinase YFG is lacZ or GFP, promoter is general and recombinase is tissue-specific Precise control of YFG gene expression -YFG is only expressed in which promoter is active and recombinase has also been expressed

43. Above + Promoter 3 Gal80 but NOT where Promoter 3 is expressed Combinatorial Approaches: Boolean Logic of Gene Expression UAS STOP YFG FRT Switch-Gal4 Promoter 1 Promoter 2 FLPase YFG will be expressed wherever Promoter 1 is active AND when hormone (RU486) is supplied, AND only in cells that have had Promoter 2 active sometime in their history Similar combinations possible with CRE, inducible CRE, TET ON and OFF, LexA, qUAS, etc.

44. Cross to: UAS-CD8-GFP--cell surface GFP Visualize neuronal connections of circuit Example: studying the neuronal circuitry driving sexual behavior in Drosophila fruitless (fru): transcription factor controlling sex-specific neuronal circuits fru-Gal4 knock in Gal4 now expressed where FRU expressed UAS-shibireTS--temperature sensitive dominant blocker of endocytosis Conditional block of neuronal function in fru circuit fru-Gal4 + UAS-FRT-STOP-FRT shibireTS + eyeless-FLP Only express shibireTS whereBOTH fru and eyeless are expressed Conditional knockout of a SUBSET of fru neurons