Genome Engineering

Question 1

How can we find out what does gene x do?

Answer 1

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

Question 2

Why is genome engineering useful?

Answer 2

Test(and utilize) functions of non-coding DNA
Where is the promoter and enhancer expressed?
Potential for gene therapy

Question 3

Transgenes

Answer 3

transfer genes between organism (genetically modified organisms) to see of there is an observed phenotype

Question 4

Gene therapy

Answer 4

Could repair mutations that cause human diseases or help the immune system fight cancer - requires transgenes or replacement of endogenous locus

Question 5

Restriction Enzymes

Answer 5

Recombinant DNA 1972

Saftey Guidelines 1975

Question 6

Gene targeting

Answer 6

Depends on homologous recombination, which is rare

1989 first knockout mouse

Question 7

inducible recombination-two common enzymes

Answer 7

Cre recombinase binds loxP sites

flippase(Flp) binds FRT sites

Question 8

inducible recombination-specificity

Answer 8

spatial : cell-type specific promoter
temporal: inducible promoter or control of nuclear localization
:Heat shock promoter i

Question 9

Cre-ER

Answer 9

restricted to the cytoplasm until binding tamoxifen

Question 10

change in expression

Answer 10

disrupt function by removing critical eons
turn genes on by removing a stop codon
swap expression cassettes?

Question 11

Cre-Lox

Answer 11

Used to build “Brainbow”

There are several variant Lox sites and recombination only occurs between identical sites

Question 12

improving efficiency of genome engineering

Answer 12

dont rely on rare events

Create a targeted double-strand break

Question 13

Trigger non-homologous End Joining

Answer 13

NHEJ doesn’t use repair template and can create small insertions or deletions

nuclease –>ends degraded –> NHEJ

Question 14

Homology-Directed repair

Answer 14

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

Question 15

How to create a double stranded break

Answer 15

identify unique cleavage site

• Zinc Finger Nucleases(ZFNs)
• TALENs (TAL effector nucleases)
• CRISPR/Cas9

Question 16

Zinc finger nucleases(ZFNs)

Answer 16

Fuse FokI restriction endonuclease to a specially designed zinc finger DNA binding domain
The dimerization activates the enzyme

Question 17

Caveat with ZFNs

Answer 17

Zinc finger DNA binding specificity may depend on context

• may have to test seq recognition
• consortium ID’d ZFs that fxn independently

Question 18

TALENs

Answer 18

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

Question 19

Caveat of TALENs

Answer 19

it takes about a week, and validation can be very tricky

Question 20

CRISPR/Cas9

Answer 20

Clustered, Regularly Interspaced, Short Palindromic Repeat/CRISPR-Associated-9
-Endonuclease with RNA guide molecule
Bacterial adaptive immunity?

Question 21

Pros about CRISPR/Cas9

Answer 21

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

Question 22

Cas9

Answer 22

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

Question 23

Crispr/Cas9 example

Answer 23

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

Question 24

Using Cas9 to target other proteins to specific regions of the genome

Answer 24

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).

Question 25

Fusing KRAB domain

Answer 25

repress transcription

using CRISPRi or CRISPR-off

Question 26

Fuse a VP64 domain

Answer 26

activate transcription using CRISPRa

Question 27

LITE

Answer 27

Light-inducible transcriptional effectors. Two proteins that inexact in blue light

Question 28

Fuse to GFP

Answer 28

Visualize nuclear location of a DNA region

Question 29

Fuse to chromatin modifying enzymes

Answer 29

to make epigenetic changes

epiCas9s

Question 30

Fuse an affinity tag

Answer 30

for region-specific chromatic immunoprecipitation: enChIP

Question 31

synthetic genomes

Answer 31

Genomic DNA is synthesized outside the organism

e

Question 32

Group led by Venter synthesizes 1.08 MB Mycoplasma mycoides genome

Answer 32

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

Question 33

“Clean genome”

Answer 33

E.Coli lacks transposable elements, psuedogenes, and phages

Question 34

Synthetic chromosomes

Answer 34

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”

Question 35

International Consortium

Answer 35

plans to do the whole yeast genome in 5 years (synthetic)

Question 36

How to build a synthetic chromosome

Answer 36

Step 1: Synthesize building blocks (BBs) from oligonucleotides
Step 2: Assemble 2-4kb mini chunks
Step 3: Replace Native III WITH MINICHUNKS

Question 37

Genome-wide binding of the CRISPR endonuclease Cas9

Answer 37

1. 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
2. Seed seq influence sgRNA abundance and specificity
3. Indel frequencies at on-target sites and 295 off-target sites