Introduction and History of Genome Modification. Adam Clore, PhD Director, Synthetic Biology Design

Transcription

1 Introduction and History of Genome Modification Adam Clore, PhD Director, Synthetic Biology Design

2 Early Non-site Directed Genome Modification

3 Homologous recombination in yeast TARGET GENE 5 Arm URA3 3 Arm Natural hyper-recombination in yeast allows for efficient recombination with only 45 bp of homology to target

4 Homologous Recombination in Mice Discovered in 1990s 2007 Capecchi, Evans and Smithies receive the Nobel Prize for medicine Suffers from poor efficiency (1 in 10^4 to 10 ^ 7 cells is recombined Requires ~1000 bp Homology Arms

5 Recombineering in Bacteria Modifying region of interest Recombination-mediated genetic engineering Incorporates Lambda Recombination Genes in controlled by an inducible promoter to allow for hyperrecombination. Requires only 50 bp of homology

6 Phage Lambda Red Genes Lambda alpha 5-3 Exonuclease Lambda beta ssdna Binding Protein, facilitates recombination Lambda Gamma Inhibits bacterial RecB/C/D complex Decreases degradation of donor Nat Protoc. 2009;4(2): doi: /nprot

7 Disrupting Genes with Double Stranded breaks 1989 Jim Habor s Lab shows that the creation of double stranded breaks greatly increases the HR efficiency in the location of the double stranded break in Yeast Similar results were seen in mammalian cells Led to the idea that targeting DSBs could induce recombination at specific sites

8 Repair of Double stranded Breaks In most Eukaryotes Non Homologous End Joining (NHEJ) is the most efficient DBS repair pathway Error prone, often creates INDELs The presence of homologous template (aka Donar DNA ) can induce recombination Efficiency and length of homology arms varies from cell line to cell line

9 MegaNucleases Naturally occurring restriction endonucleases are great if you want to cut the bp site they recognize. Marcaida M J et al. PNAS 2008;105:

10 Zinc Finger Nucleases First generation of enzymes capable of targeting specific DNA sequences of interest. Zinc Finger domains were first observed in transcription factors found in Xenopus Best characterized are the Cys 2 His 2 class Often occur in tandem binding sequential sequences of DNA

11 Zinc Finger Nucleases The addition of the FokI Nuclease domain gives zinc fingers the ability to cleave DNA in a double stranded manner FokI requires dimerization to cleave, separate zinc finger proteins fused to FokI monomers requires two binding events for cleavage. Nature Biotechnology 25, (2007)

12 Limitations to ZFNs Unpredictable specificity While ZF domains corresponding to all 64 codons have been identified the modularity is somewhat unpredictable and off target effects are common Complex design and construction Construction of ZFNs requires 2-4 motifs per subunit, with a high degree of repeats High failure rates Limited application for high throughput screening

13 Second Generation programmable nucleases-talens Transcription Activator-Like Effectors Proteins that mimic plant transcription regulators in plants Used by Xanthomonads to effect the gene regulation of their host plants Modifies the plants gene expression to facilitate bacterial infection and deregulate hypersensitive response

14 TALEs structure Conserved repeat elements with hypervariable 12 th and 13 th aa in each alpha helix Multiple NLSs and AD domain

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16 TALEs Code NI = A HD = C NG = T NN = G or A NK = G *less effective than NN if used alone

17 TALENS-Transcription Activator-Like Effector Nucleases a TAL effector DNA binding domain + DNA cleavage domain (usually FokI)

18 Limitations to TALENs repeats of identification motifs are very challenging to construct and often unstable when cloned Construction of the 2-3 kb of coding sequence per target further limits high throughput screens, creates costly and time consuming experiments

19 CRISPR Easy Genome Modification Clustered Regularly Interspaced Short Palindromic Repeat A prokaryotic defense mechanism that screens for and cleaves specific DNA sequences Can be used to create targeted changes to the genomes of bacteria, archaea, and eukaryotes

20 The 3 Stages of CRISPR Resistance Stage 1: CRISPR Adaptation Foreign DNA is incorporated in the CRISPR array. Stage 2: CRISPR Expression CRISPR RNAs (crrnas) are transcribed from CRISPR locus. Stage 3: CRISPR Interference Foreign nucleic acid complementary to the crrna is neutralized.

21 Utilizing CRISPR for Genome Modification We need 3 components: 1. CRISPR Associated Gene 9 (CAS9) 2. RNA with CRISPR repeats (crrna) 3. Trans-acting RNA (tracrrna) * 2 and 3 can be combined into a single sequence called a single guide RNA (sgrna) Zhang lab:

22 Overview of the three generations of programmable nucleases Zinc Finger Nucleases Cost Reliability Accuracy $$$$ Low Poor TALENs $$$$ High Good CRISPR $ High Good

23 gblocks Gene Fragments for CRISPR

24 gblocks Gene Fragments for CRISPR