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Engineering Programmable Nucleases: Applications in the Study of Gene Function and eventually Gene Therapy Scot Wolfe Molecular, Cell and Cancer Biology UMass Medical School Frontiers in Science Presentation March 18th, 2015 One of our fundamental areas of exploration is the development of tools for the targeted engineering of genomes. One of our fundamental areas of exploration is the development of tools for the targeted engineering of genomes. In analogy with surgical tools - we endeavor to make genomic scalpels that permit the precise modification of the genome. One of our fundamental areas of exploration is the development of tools for the targeted engineering of genomes. In analogy with surgical tools - we endeavor to make genomic scalpels that permit the precise modification of the genome. These tools have utility for: 1) The study of gene function during vertebrate development 2) The correction of disease-causing genetic lesions How do we locate a specific “address” within the genome? Gene X How do we locate a specific “address” within the genome? Gene X How do we locate a specific “address” within the genome? Gene X -TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA-AGCAAGCCGAGCGCAATTCGTTCACGTCTT- How do we locate a specific “address” within the genome? Gene X -TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA-AGCAAGCCGAGCGCAATTCGTTCACGTCTTSequence-specific DNA recognition module How do we locate a specific “address” within the genome? Gene X -TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA-AGCAAGCCGAGCGCAATTCGTTCACGTCTTSequence-specific DNA recognition module Where do we begin? - By understanding how naturally-occurring DNA-binding proteins locate their sequences. Cys2His2 Zinc Finger proteins (ZFPs) are the most diverse family of DNA-binding domains in vertebrates Cys2His2 Zinc Finger proteins (ZFPs) are the most diverse family of DNA-binding domains in vertebrates How can we understand DNA recognition by these proteins? A bacterial one-hybrid system can be used to determine DNA-binding specificity RNA polymerase Zinc Finger Protein ZFP Weak Promoter Reporter Vector Bacterial Selection Strain ΔrpoZ ΔhisB ΔpyrF A bacterial one-hybrid system can be used to determine DNA-binding specificity RNA polymerase Zinc Finger Protein ZFP Match Reporter Vector Bacterial Selection Strain ΔrpoZ ΔhisB ΔpyrF Weak Promoter A bacterial one-hybrid system can be used to determine DNA-binding specificity RNA polymerase Zinc Finger Protein ZFP Match Weak Promoter Reporter Vector Bacterial Selection Strain ΔrpoZ ΔhisB ΔpyrF ZFP ZFP ZFP ZFP Noyes et al. Nucleic Acids Research, 2008, 36, 2547-2560. DNA binding site selections are performed in a single round to yield interacting partners Overview of a B1H binding site selection pol-ZFP DNA binding site selections are performed in a single round to yield interacting partners Overview of a B1H binding site selection pol-ZFP DNA binding site selections are performed in a single round to yield interacting partners Overview of a B1H binding site selection pol-ZFP DNA binding site selections are performed in a single round to yield interacting partners Overview of a B1H binding site selection pol-ZFP SP & KLF M4 ZFA sr lo l p h a -P O o CG3065 F1-5 1 4 ph 6 8 ol lol PL a- A P 2 al 78 lo 31 G C 19 3 8 CG 9B D1 f vl PN lolaY lola-P 71 CG110 lola-PK Cf2-PB kr ttk-PA pad lolaPD crolF716 CG 736 8 l(3) neo CG 38 sa 1 2 2 3 lr 6 -P ttk -F3B 5 ab -PF -P B 5-7 peb-F o vo fru PE r bPC b rPL b rhb C ZI A SN T CR &S br -P A -P F od d so b bo w l lola -PU lola -PT la lola-PJ lola-PW chinmo peb-F1-3 36-PA lola-PQ CG122 CG14 her 962 lo wor 7181 CG1 sn a SP AL T d lm g su a op 3 -5 ci g F 05 es 6 12 CG t scr E -P 7 ab 26 10 CG & O S D k ipp D ed T Cf2-PA lola-PP HN OVO ZEP CG7928 shn-F1-2 Aef1 LI G DIS CO p- FEZ PC 2 ke P P I D1 G 9A se ns 2 sen disc s o -rF12 disc o CG3 407 CG31 670 CG4854 CG4360-F1-3 a- gl n GF I a- a- sq z rn 52 20 C G 1 -9 -F jim lol lol lol 0 YY 1 CG5669 Sp1 btd bteb2 hkb CG 120 29 CG 9 lun 895 a klu BL IM P1 EG R im Bl We have determined the DNAbinding specificity of more than 100 naturallyoccurring zinc finger proteins (ZFPs) and many more synthetic ZFPs Enuameh, et al. Genome Research 2013 SP & KLF M4 ZFA sr lo l p h a -P O o CG3065 F1-5 3 2 1 lola-PJ lola-PW chinmo peb-F1-3 36-PA lola-PQ CG122 CG14 wor 7181 CG1 sn a 3’ her 962 lo la M 3 DNA triplet 3’ SP AL T br -P A -P F od d so b bo w l lola -PU lola -PT 5’ 5 kr ttk-PA pad lolaPD crolF716 CG 736 8 l(3) neo CG 38 sa 1 2 2 3 lr 6 -P ttk -F3B 5 ab -PF -P B d lm g su a op 3 -5 ci g F 05 es 6 12 CG t scr E -P 7 ab 26 10 CG PL a- A P 2 al 78 lo 31 G C 19 3 8 CG 9B D1 f vl PN lolaY lola-P 71 CG110 lola-PK CG7928 shn-F1-2 5’ T CR &S Cf2-PA lola-PP 5-7 peb-F o vo fru PE r bPC b rPL b rhb -1 lol ZEP 6 5 ol A SN 8 Cf2-PB O S D k ipp D ed T ph 6 Aef1 HN OVO CG5669 Sp1 btd bteb2 hkb CG 120 29 CG 9 lun 895 a klu 4 C ZI GF I 2 1 DIS CO p- n FEZ PC ke P P I D1 G 9A se ns 2 sen disc s o -rF12 disc o CG3 407 CG31 670 CG4854 CG4360-F1-3 a- gl Zn & a- a- sq z rn 52 20 C G 1 -9 -F jim lol lol lol 0 YY 1 LI G BL IM P1 EG R im Bl We have determined the DNAbinding specificity of more than 100 naturallyoccurring zinc finger proteins (ZFPs) and many more synthetic ZFPs Enuameh, et al. Genome Research 2013 We have constructed an archive of characterized zinc finger modules can be assembled into ZFPs to recognize a specific “address” within the genome Two Finger modules) ZincArchive Finger (194 Nucleases OneC Finger Archive (27 modules) We have constructed an archive of characterized zinc finger modules can be assembled into ZFPs to recognize a specific “address” within the genome Two Finger modules) ZincArchive Finger (194 Nucleases OneC Finger Archive (27 modules) -TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA1 2 3 1 2 3 -AGCAAGCCGAGCGCAATTCGTTCACGTCTT- We have constructed an archive of characterized zinc finger modules can be assembled into ZFPs to recognize a specific “address” within the genome Two Finger modules) ZincArchive Finger (194 Nucleases OneC Finger Archive (27 modules) aZFP -TCGTTCGGCTCGCGTTAAGCAAGTGCAGAA1 2 3 1 2 3 -AGCAAGCCGAGCGCAATTCGTTCACGTCTT- ZFPs or other platforms can be used to create artificial nucleases that allow targeted gene editing ZFPs or other platforms can be used to create artificial nucleases that allow targeted gene editing ZFPs or other platforms can be used to create artificial nucleases that allow targeted gene editing ZFPs or other platforms can be used to create artificial nucleases that allow targeted gene editing Zinc Finger Nuclease construction of disease models in zebrafish Diabetes: leptin receptor (db) miR-375 (1 & 2) Obesity: melanocortin receptor 4 (mc4r) melanocortin receptor 3 (mc3r) leptin (A & B) (ob) Ankit Gupta, Victoria Hall, Dana DeSantis Zinc Finger Nuclease construction of disease models in zebrafish Diabetes: leptin receptor (db) miR-375 (1 & 2) Obesity: melanocortin receptor 4 (mc4r) melanocortin receptor 3 (mc3r) leptin (A & B) (ob) Ankit Gupta, Victoria Hall, Dana DeSantis Pancreatic islet development is largely conserved between humans and zebrafish 6 hpf 10 hpf 11 hpf 12 hpf 14 hpf 15 hpf Tiso et al., Mol. Cell Endocrinology 312, 24-30, 2009 miRNAs can inhibit translation and destabilize their target mRNAs miR-375 Winter et. al., Nature Cell Bio., 2009,11(3):228-34 Fo kI Two Copies of miR-375 are present in the zebrafish genome Spacer (6bp) ~ ~ genomic target site (mir-375-2) Fo kI Fo kI Spacer (6bp) zebrafish miR-375-2 pri-miRNA hairpin I Fo k donor plasmid 60 50 inverted A A G A G G G U U site U U G U U C G U U C G G C U C U U A C 80 G C G U G C A G A U G CA GACGU C C G U A A C A C G C A C A C C G A G U U G C A U G U U C A U GU 30 NHEJ-mediated ligation Fo kI ZFN target site C U C 5’ ZFP site 10 3’ ZFP site Spacer (6bp) Spacer (6bp) Gal4FF miR375-2 TCGTTCGGCTCGcgttacGCAGATGCAGAC CGAGCCGAACGA GCAGATGCAGAC Fo kI CGAGCCGAACGA GCAAGTGCAGAA Fo kI TCGTTCGGCTCGcgttaaGCAAGTGCAGAA ~ ~ genomic target site miR375-1 (mir-375-2) 20 Fo kI A 70 Gal4FF Ankit Gupta Injecting nucleases into zebrafish embryos cap pCS-HA-ZFN SP6 NLS HA 1 2 3 Fok I DD cap pCS-Flag-ZFN SP6 polyA signal polyA signal NLS inject both ZFN mRNAs into 1-cell stage embryos FL 1 2 3 Fok I RR Fo kI ObLiGaRe: precise NHEJ-mediate ligation of exogenous DNA sequences within the genome Spacer (6bp) ~ ~ genomic target site (mir-375-2) Fo kI Fo kI Spacer (6bp) Gal4FF I Fo k donor plasmid inverted site Fo kI Spacer (6bp) Spacer (6bp) Fo kI Fo kI Maresca, et. al. (2013). Genome Research, 23(3), 539. ~ Gal4FF ~ genomic target site (mir-375-2) Fo kI NHEJ-mediated ligation Fo kI ObLiGaRe: precise NHEJ-mediate ligation of exogenous DNA sequences within the genome Spacer (6bp) ~ ~ genomic target site (mir-375-2) Fo kI Fo kI Spacer (6bp) Gal4FF I Fo k donor plasmid inverted site Fo kI Spacer (6bp) Spacer (6bp) Fo kI Fo kI Maresca, et. al. (2013). Genome Research, 23(3), 539. ~ Gal4FF ~ genomic target site (mir-375-2) Fo kI NHEJ-mediated ligation Fo kI ObLiGaRe: precise NHEJ-mediate ligation of exogenous DNA sequences within the genome Spacer (6bp) ~ ~ genomic target site (mir-375-2) Fo kI Fo kI Spacer (6bp) Gal4FF I Fo k donor plasmid inverted site Fo kI Spacer (6bp) Spacer (6bp) Fo kI Fo kI Maresca, et. al. (2013). Genome Research, 23(3), 539. ~ Gal4FF ~ genomic target site (mir-375-2) Fo kI NHEJ-mediated ligation Fo kI ObLiGaRe: precise NHEJ-mediate ligation of exogenous DNA sequences within the genome Spacer (6bp) ~ ~ genomic target site (mir-375-2) Fo kI Fo kI Spacer (6bp) Gal4FF I Fo k donor plasmid inverted site Fo kI Spacer (6bp) Spacer (6bp) Fo kI Fo kI Maresca, et. al. (2013). Genome Research, 23(3), 539. ~ Gal4FF ~ genomic target site (mir-375-2) Fo kI NHEJ-mediated ligation ObLiGaRe insertion into mir-375-2 locus Fo kI Fo kI 5’ primer ~ Gal4FF Fo kI Fo kI 3’ primer embryos (N/D) N N N N D N D ZFNs (pg) 0 63 0 63 63 125 125 Donor (pg) 0 0 25 25 25 25 25 ladder ~ genomic target site (mir-375-2) 1.3% Ankit Gupta miR-375-2 knockin fish crossed to insulin:mcherry UAS:EGFP Pancreas Pituitary ins:mcherry overlay miR-375-2 knockin fish allows pancreatic progenitor cells to be followed during development (23 to 30 hpf) Ankit Gupta miR-375-2 knockin fish allows pancreatic progenitor cells to be followed during development (23 to 30 hpf) Ankit Gupta A subset of miR-375 positive cells express insulin at 2 dpf miR-375-2 Gal4FF; UAS: EGFP ins: mcherry overlay miR-375-1-/- miR-375-2 -/- animals display defects in pancreatic islet formation 3 dpf - insulin:mCherry WT double mutant miR-375 null animals have reduced β-cell numbers at 3dpf -cell number at 3 dpf 30 p<0.001 27 16 20 10 N ul ls 0 W T insulin:mCherry cells 40 miR-375 Genotype Ankit Gupta miR-375 nulls show elevated glucose levels at 6 dpf p<0.001 36 M 40 20 18 M ul ls N T 0 W whole embryo glucose concentration 60 miR-375 Genotype Ankit Gupta miR-375 positive cells can be sorted from wild-type and mutant animals to identify genes that are potentially responsible for the observed phenotypes miR-375-2 Gal4FF; UAS: EGFP ins: mcherry overlay Fold Change relative to WT Upregulated genes that are potential miR-375 targets 1000 104.0 100 147.0 10 4.3 beta cell alpha cell 6.1 5.3 3.5 1 ELAVL4 ELAVL3 Ebf3 The CRISPR/Cas9 system provides a simple system for genome editing CRISPR/Cas9 system provides RNA guided nuclease activity -3’ -5’ S. pyogenes Cas9 (SpCas9) : nGG >> nAG; nGA >> others The primary constraint on target recognition is the PAM specificity of the Cas9 protein Jinek et al. Science. 2012; 337(6096):816-21 Cong et al. Science. 2013; 339(6121):819-23 Development of chimeric Cas9 - DNAbinding domain (DBD) fusion Hypothesis: Fusion of Cas9 to a programmable DNA-binding domain will provide improved genome editing precision and broader targeting range. Target site -3’ -5’ DBD Development of chimeric Cas9 - DNAbinding domain (DBD) fusion Hypothesis: Fusion of Cas9 to a programmable DNA-binding domain will provide improved genome editing precision and broader targeting range. Target site -3’ -5’ DBD Goals:1) Identify parameters to create functional Cas9-DBD framework 2) Attenuate Cas9 to make nuclease activity DBD dependent GFP reporter assay to monitor nuclease activity in cell culture mammalian cell homology driven repair with functional nuclease Wilson et al. MTNA (2013) 2, e87 GFP reporter assay to monitor nuclease activity in cell culture mammalian cell homology driven repair with functional nuclease Wilson et al. MTNA (2013) 2, e87 GFP reporter assay to monitor nuclease activity in cell culture mammalian cell homology driven repair with functional nuclease Wilson et al. MTNA (2013) 2, e87 Identifying parameters to create a functional Cas9-DBD framework Identifying parameters to create a functional Cas9-DBD framework Identifying parameters to create a functional Cas9-DBD framework A DBD fusion expands the targeting repertoire of spCas9 Cas9-ZFP is functional on genomic targets with suboptimal PAMs Cas9-ZFP = SpCas9-Zif268 T7 Endonuclease I (T7EI) assay (11bp) Cas9-ZFP is functional on genomic targets with suboptimal PAMs Cas9-ZFP = SpCas9-Zif268 T7 Endonuclease I (T7EI) assay (11bp) To attenuate independent Cas9 activity mutagenesis was performed on the PAM contact residues PAM recognition by Arg 1333 and Arg 1335 Arg1335 Arg1333 Anders et al. Nature (2014) 513, 569–573 Arg1333 and Arg1335 mutants cause loss of function which can be restored by a ZFP fusion Arg1333 Lysine Serine Arg1335 Lysine Serine Arg1333 and Arg1335 mutants cause loss of function which can be restored by a ZFP fusion Arg1333 Lysine Serine Arg1335 Lysine Serine Arg1333 and Arg1335 mutants cause loss of function which can be restored by a ZFP fusion Arg1333 Lysine Serine Arg1335 Lysine Serine Can Cas9-ZFPs improve precision? GTGGGTGAGTGAGTGTGTGCGTGTGGGGTTGAGGGCGTTGGAGCGGGG VEGF-A Cas9 TS3 ZFP target site Fu et al. Nat Biotechnol. 2014; 32(3):279-84 Can Cas9-ZFPs improve precision? GTGGGTGAGTGAGTGTGTGCGTGTGGGGTTGAGGGCGTTGGAGCGGGG VEGF-A Cas9 TS3 ZFP target site Fu et al. Nat Biotechnol. 2014; 32(3):279-84 TS3 OT3-2 Control Cas9-MT3-ZFTS3 sgRNA-TS3 Cas9-MT3 sgRNA-TS3 Cas9-WT sgRNA-TS3 Control Cas9-MT3-ZFTS4 sgRNA-TS3 Cas9-MT3-ZFTS3 sgRNA-TS4 Cas9-MT3-ZFTS3 sgRNA-TS3 Cas9-MT3 sgRNA-TS3 Cas9-WT sgRNA-TS3 Cas9-ZFPs increase the precision of genome editing TS3 OT3-2 Control Cas9-MT3-ZFTS3 sgRNA-TS3 Cas9-MT3 sgRNA-TS3 Cas9-WT sgRNA-TS3 Control Cas9-MT3-ZFTS4 sgRNA-TS3 Cas9-MT3-ZFTS3 sgRNA-TS4 Cas9-MT3-ZFTS3 sgRNA-TS3 Cas9-MT3 sgRNA-TS3 Cas9-WT sgRNA-TS3 Cas9-ZFPs increase the precision of genome editing TS3 OT3-2 Control Cas9-MT3-ZFTS3 sgRNA-TS3 Cas9-MT3 sgRNA-TS3 Cas9-WT sgRNA-TS3 Control Cas9-MT3-ZFTS4 sgRNA-TS3 Cas9-MT3-ZFTS3 sgRNA-TS4 Cas9-MT3-ZFTS3 sgRNA-TS3 Cas9-MT3 sgRNA-TS3 Cas9-WT sgRNA-TS3 Cas9-ZFPs increase the precision of genome editing Artificial nucleases are powerful tools for manipulating vertebrate genomes in a directed manner Artificial nucleases are powerful tools for manipulating vertebrate genomes in a directed manner 1) Artificial nuclease platforms have been developed that can target the majority of sequences with a vertebrate genome. Artificial nucleases are powerful tools for manipulating vertebrate genomes in a directed manner 1) Artificial nuclease platforms have been developed that can target the majority of sequences with a vertebrate genome. 2) These tools allow the study of gene function in model organisms and the creation of disease models to understand dysfunction at the systemic and molecular level Artificial nucleases are powerful tools for manipulating vertebrate genomes in a directed manner 1) Artificial nuclease platforms have been developed that can target the majority of sequences with a vertebrate genome. 2) These tools allow the study of gene function in model organisms and the creation of disease models to understand dysfunction at the systemic and molecular level 3) More precise nucleases are being developed that will permit the realization of genetic correction of aberrant loci for the treatment of disease. Acknowledgements UMass Medical School (UMMS) Molecular, Cell and Cancer Biology (MCCB) Wolfe Lab Fatih Bolukbasi Ankit Gupta Dana DeSantis Victoria Hall Selase Enuameh Cong Zhu Yuna Asriyan Amy Rayla Stephanie Chu Heather Bell Marcus Noyes Brodsky Lab •Michael Brodsky Sarah Oiekemus Bioinformatics MCCB Julie Zhu Jianhong Ou Maehr Lab (DCOE) René Maehr Nicola Kearns Ryan Genga Hannah Pham Bioinformatics Core (UMMS) Manuel Garber Alper Kucukural Pederson Lab (BMP) Thoru Pederson Hanhui Ma Nathan Lawson (MCCB) Nick Rhind (BMP) Michael Parsons (Johns Hopkins) Funding: NIGMS, NHLBI, NIAID & NHGRI Jack Leonard Karl Simin Erik Sontheimer Laura Alonso Jeremy Luban