Genome Engineering Flashcards
How can we find out what does gene x do?
by tagging the protein it encodes for and tracking it. This can be done using a transgene and/or replacing the endogenous locus. You can also break the gene and see what goes wrong (reverse genetics). Also you can expressing the gene somewhere new, using a transgene
Why is genome engineering useful?
Test(and utilize) functions of non-coding DNA
Where is the promoter and enhancer expressed?
Potential for gene therapy
Transgenes
transfer genes between organism (genetically modified organisms) to see of there is an observed phenotype
Gene therapy
Could repair mutations that cause human diseases or help the immune system fight cancer - requires transgenes or replacement of endogenous locus
Restriction Enzymes
Recombinant DNA 1972
Saftey Guidelines 1975
Gene targeting
Depends on homologous recombination, which is rare
1989 first knockout mouse
inducible recombination-two common enzymes
Cre recombinase binds loxP sites
flippase(Flp) binds FRT sites
inducible recombination-specificity
spatial : cell-type specific promoter
temporal: inducible promoter or control of nuclear localization
:Heat shock promoter i
Cre-ER
restricted to the cytoplasm until binding tamoxifen
change in expression
disrupt function by removing critical eons
turn genes on by removing a stop codon
swap expression cassettes?
Cre-Lox
Used to build “Brainbow”
There are several variant Lox sites and recombination only occurs between identical sites
improving efficiency of genome engineering
dont rely on rare events
Create a targeted double-strand break
Trigger non-homologous End Joining
NHEJ doesn’t use repair template and can create small insertions or deletions
nuclease –>ends degraded –> NHEJ
Homology-Directed repair
IF repair template is present we can introduce insertions, precise deletions, or point mutations
Oligo-based
Nuclease-> end resection–>synthetic oligo annealing–> error free insertion
Plasmid based
nuclease –>homologous recombination–>modified locus
promoted by the use of only one function FokI domain to create a nickase
How to create a double stranded break
identify unique cleavage site
- Zinc Finger Nucleases(ZFNs)
- TALENs (TAL effector nucleases)
- CRISPR/Cas9
Zinc finger nucleases(ZFNs)
Fuse FokI restriction endonuclease to a specially designed zinc finger DNA binding domain
The dimerization activates the enzyme
Caveat with ZFNs
Zinc finger DNA binding specificity may depend on context
- may have to test seq recognition
- consortium ID’d ZFs that fxn independently
TALENs
Transcription Activator-Like Effector Nucleases
TAL effectors secreted by Xathomonas bacteria + FokI nuclease
DNA binding domain: one repeat of 33-35 AA binds one base
Caveat of TALENs
it takes about a week, and validation can be very tricky
CRISPR/Cas9
Clustered, Regularly Interspaced, Short Palindromic Repeat/CRISPR-Associated-9
-Endonuclease with RNA guide molecule
Bacterial adaptive immunity?
Pros about CRISPR/Cas9
does not require creation of a large synthetic peptide
only the sgRNA (20 bp) has to be altered to target a different site in the genome
easy to customize for new targets
Cas9
RNA guided endonuclease
two determinants of specificity
-complementarity of target DNA and sgRNA
-Binding to the PAM site just upstream of the target sequence ( NGG for S. progenies Cas9)
-minimal seq requirements for target cleavage
Crispr/Cas9 example
The ciliary protein CHE-12
has 4 microtubule binding TOG domains. CHE-12 binds microtubles and promoters MT polymerization in vitro
localizes to the primary cilium in transfected cells
Questions
Is CHE-12 important for cilia formation in vivo? Knockout mutant. Where does it localize? GFP-tagged knocking. Is microtubule binding important for CHE-12 function?Targeted mutations in the endogenous locus
Using Cas9 to target other proteins to specific regions of the genome
Use catalytically dead form of Cas9 that can’t cut DNA (can also use TALEs or ZFs). This is used to alter transcription.
Un-tagged to block transcriptional elongation (not as effective in eukaryotes).
Fusing KRAB domain
repress transcription
using CRISPRi or CRISPR-off
Fuse a VP64 domain
activate transcription using CRISPRa
LITE
Light-inducible transcriptional effectors. Two proteins that inexact in blue light
Fuse to GFP
Visualize nuclear location of a DNA region
Fuse to chromatin modifying enzymes
to make epigenetic changes
epiCas9s
Fuse an affinity tag
for region-specific chromatic immunoprecipitation: enChIP
synthetic genomes
Genomic DNA is synthesized outside the organism
e
Group led by Venter synthesizes 1.08 MB Mycoplasma mycoides genome
Included ‘watermarks’ designed deletions as well as errors generated by the synthesis process
1kb synthesis, ligation and joining by multiplex PCR
sufficient to replace an existing cell’s genome
“Clean genome”
E.Coli lacks transposable elements, psuedogenes, and phages
Synthetic chromosomes
Designer S.cerevisiae chromosome III, 2.5% of genome “Build a Genome” project at JHU
Replace small chunks of the endogenous chromosome by homologous recombination
changed ~50kb
removed transposons and introns
replaced all UAG stops with UAA to free up codon
included loxPsym sites to allow future “genome scrambling”
International Consortium
plans to do the whole yeast genome in 5 years (synthetic)
How to build a synthetic chromosome
Step 1: Synthesize building blocks (BBs) from oligonucleotides
Step 2: Assemble 2-4kb mini chunks
Step 3: Replace Native III WITH MINICHUNKS
Genome-wide binding of the CRISPR endonuclease Cas9
- Genome-wide in vivo binding of dCas9-sgRNA
integration via piggyBac, transfection, HA-ChIP
2.A 5-nucleotide seed for dCas9 binding
3.Chromatin accessibility is a major determinant of binding in vivo - Seed seq influence sgRNA abundance and specificity
- Indel frequencies at on-target sites and 295 off-target sites
