Gene editing and engineering

Question 1

Why is the mouse used as a genetic model? What are its limitations?

Answer 1

-Share virtually the same set of genes as humans
-Protein-coding regions of mouse and human genomes are very similar, but less the case for non-coding regions
-Possible to test the function of genes in mice

Limitations:
-Different cell duplication time, lifespan and cancer susceptibility than human
-Limited translational impact for neurodegenerative diseases

Question 2

What is gene targeting through homologous mediated recombination in mouse ESCs? How does it work?

Answer 2

Embryonic stem cells (ESCs) are cultured and electroporated to introduce a targeting vector.

Once a pure population of targeted ESC is obtained (via pos-neg selection, see below), the culture is injected into blastocysts, which are then implanted into foster mothers.

Works via a targeting vector, which contains:

-3’ and 5’ homology prime to recognize genomic region of interest
-Gene of interest (point mutation if goal is to KO good copy)
-A neo-gene that can confer resistance (e.g., chemical) -> will allow to get rid of all the cells that do not express this protective agent = purifying the cell population expressing the genetic construct = POSITIVE selectivity
-A neg. sel. gene that allows to induce apoptosis to all the cells expressing that gene, which occurs only if the desired genetic modification did not work as intended (vector not perfectly integrated in host cells) = NEGATIVE selectivity

Targeting vector must be introduced in the cell at phase of synthesis for homologous recombination to happen.

Detailed steps of cross-strain gene editing process via using HR and ESC:

1. Culturing ESCs from the donor blastocyst from a mouse line with fur color A.
2. Introduction of targeting vector in ESC culture.
3. Enriching ESC population with gene construct via neg-pos selection.
4. Picking of resistant clones.
5. DNA preparations from ESC clones are analyzed via southern blot
6. Clones positive for the gene modification are isolated and given time to reproduce.
7. HR ESC clones are injected in a recipient blastocyst from a mouse line with fur color B.
8. Blastocyst surgically transferred to foster albino mother.
9. Select pups with fur color A and further breed them with WT to establish a model line.

Question 3

What is the main limitation of using gene editing techniques that require to occur during the development of the animal model?

Answer 3

Genetically engineered mutations that are homozygous can prohibit functional characterization and cause early developmental confounds.

Question 4

What are the two main advantages of cre-lox?

Answer 4

Can be spatially (tissue) and temporally restricted.

Question 5

What are the two mouse lines required for cre-lox?

Answer 5

One line that is homozygous for a gene flanked by loxP sites

One line that is heterozygous for a gene flanked by loxP sites + that expresses Cre

Question 6

What are the three main genetic modifications that can mediate Cre-Lox?

Answer 6

Excision
Inversion
Translocation

Question 7

What is an alternative to the Cre-Lox system?

Answer 7

FLP-FRT recombination, works similarly to Cre (also uses FloxP sites), less used bc. temperature sensitivity

Question 8

What is CreER-Lox recombination?

Answer 8

Cre is fused to an estrogen receptor -> prevents localization to the nucleus until administration of tamoxifen, which displaces Cre, allowing it to enter the nucleus and undertake recombination

Question 9

Does HR targeting ES cells work in higher mammalian model organisms?

Answer 9

No

Question 10

What is an alternative way to edit the genome of higher mammalian model organisms?

Answer 10

Zinc-Finger Nucleases

Question 11

How does targeted genome modification through zinc-finger nucleases work?

Answer 11

Directly injecting zinc-finger nucleases into the fertilized egg -> no need for cell culture

Zinc-finger nucleases = zinc-finger domains are designed to bind a DNA region of interest and are fused to Fok1 nuclease domains that dimerize to excise targeted DNA segment

Question 12

What are type II restriction enzymes and for what purpose are they often used?

Answer 12

Molecular scissors that cleave DNA, used to insert DNA segments in plasmids

Question 13

What confers specificity to ZFNs?

Answer 13

An array of zinc finger proteins (ZFN finger domain) can be composed of specific fingers to bind a DNA segment of interest

Question 14

How can non-homologous end joining and ZFNs be coupled?

Answer 14

ZFNs can be used to induce a double-strand break at a specific location, while NHEJ will fuse the ends of the severed DNA segments while introducing insertion/deletions, effectively knocking-out the gene of interest

Question 15

What is TALE? What is TALEN? How does it work?

Answer 15

Transcription Activator-Like Effector

Composed of an array of monomers, each binding a single nucleotide = high specificity

TALEN = TALE fused to a nuclease, allowing to induce a double-strand break at a specific location

Question 16

Why is TALE more specific than ZFN?

Answer 16

Zinc fingers bind 3 nucleotides at the time, while TALE monomers bind 1 nucleotide at the time

Question 17

What is a surveyor nuclease assay? What is the objective?

Answer 17

TALEN cut ->error prone DNA NHEJ -> PCR -> hybridization (formation of heteroduplexes), necessary for surveyor to bind properly -> selective cut of heteroduplexes with surveyor nuclease (enzyme recognizes all base substitutions and insertions/deletions, and cleaves the 3′ side of mismatched sites in both DNA strands with high specificity) -> quantification of PCR products with electrophoresis, presence of DNA fragments = TALEN cut was successful = specific

Question 18

What are limitations to targeted genome editing with ZFN and TALEN? What is the most common and recent alternative system?

Answer 18

Both systems are based on DNA/protein interaction ->on-target and off-target prediction/validation difficult + sensitive to epigenetic status of targeted sequence + time ineffective

CRISPR/Cas9

Question 19

How does the bacterial CRISPR-Cas9 system work?

Answer 19

CRISPR-Cas9 system in naturally encoded in some bacterial genomes

CRISPR (clustered regularly interspaced short palindromic repeats) RNA (crRNA) = array of DNA sequences (spacers) matching the DNA of bacteriophages or other pathogens to the bacteria, spaced by repeat sequences, which play a role in the processing of precursor CRISPR RNA (pre-crRNA) into individual mature crRNAs.

// In bacteria and archaea; CRISPR-Cas (clustered, regularly interspaced short palindromic repeats/CRISPR-associated proteins) constitute an RNA-mediated defense system which protects against viruses and plasmids. This defensive pathway has three steps. First a copy of the invading nucleic acid is integrated into the CRISPR locus. Next, CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus. The crRNAs are then incorporated into effector complexes, where the crRNA guides the complex to the invading nucleic acid and the Cas proteins degrade this nucleic acid.

Question 20

How can CRISPR-Cas9 mutate multiple genes in one step?

Answer 20

Introduction of several gRNAs

Question 21

What is the advantage of double nicking by RNA-guided CRISPR Cas9?

Answer 21

Enhanced specificity (avoid off-target effects)

Notably used with Fok1 -> fCas9 (x2 inactive Cas9 allowing for specific targeting)

Question 22

What is a significant advantage of SaCas9 compared to SpCas9?

Answer 22

SaCas9 is smaller, allowing to be packaged in AAV vectors = can be delivered in-vivo

Question 23

What is the PAM sequence?

Answer 23

The protospacer adjacent motif (PAM) is a DNA sequence usually 3-4 nucleotides downstream to the DNA sequence targeted by Cas9, allowing the latter to cleave the DNA strand

Different Cas9s recognized different PAMs -> the shorter the recognized sequence, the more flexibility (and less specificity)