BIOL 3000 CRISPR/Cas 9 Gene Editing

Innate immunity

What you are born with

Surface barriers, cellular barriers, and inflammation

Acquired immunity

Comes from when you get an infection

Lymphocytes, T-Cells, B-Cells

Immunological Memory

The memory of getting an infection and fighting it

Bacterial immunity

Bacteriophages (“virus of bacteria”) that attack our cells, using an almost needle-like structure.

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

Short palindromic repeats

Forms hairpin-like structures that are regularly spaced

Clustered regularly interspaced

Spacer DNA that is completely unique in sequence

Each matches up perfectly with a viral DNA (bacteriophage)

CRISPR region

Includes repeats of palindromic DNA sequences and unique spacers

CAs genes

CRISPR Associated Genes: helicases and endonucleases

Helicases

Unwinds the DNA

Endonucleases

Cuts the DNA

Summarize the typical bacteriophage infection

1. Bacteriophage injects its DNA

2. It then works and makes viral DNA

3. The cell now contains many viral capsules that are continuously copied and made

4. Cell dies

Summarize bacteriophage infection with CRISPR

1. The bacteriophage injects its DNA

2. If the CRISPR is there, then the CAs proteins are made

3. These proteins then come in with CRISPR RNA and the RNA is able to bind to the CRISPR because it is an exact copy

   1. The endonucleases then eat up viral DNA

Palindromic repeats

tracrRNA creates a hairpin-like structure

Protosequences

crRNA

Analogous to CRISPR RNA and is a complement to some region of a DNA sequence

guideRNA (gRNA)

The structure of protosequences and tracrRNA

Cas9

Made of Streptococcus pyogenese

Does both helicase and endonuclease activities by itself

Control CRISPR/Cas9 Plasmid

A circular plasmid that contains the U6 promoter, 20 nt sequence, gRNA, termination signal, CBh promoter, nuclear localization signal, SpCas9 ribonuclease, 2A peptide, and sometimes the Green Fluorescent Protein (GFP)

Why is the CRISPR/Cas9 plasmid circular?

So endonucleases can’t eat it up

U6 promoter

Drives the expression of gRNA, in front of the gRNA

2A peptide

Allows the production of both Cas9 and GFP from the same CBh promoter

How does CRISPR/Cas9 work?

1. Cas9 opens up and looks for the Protospacer Adjacent Motif (PAM)- 5’-NGG-3’. Once found, PAM binds to it.

2. Then endonucleases cuts out PAM

3. Two ways of double-stranded repairs are possible: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR)

Non-Homologous End Joining (NHEJ)

The end of the DNA strands are directly ligated back together. Often resulting in insertions or deletions. Used to Knock-Out genes

Homologous Recombination (HR)

A DNA template is added to the mix with sequence matching the areas flanking the target site. This allows precise mutations to be inserted. Used in Knock-In genes

CRISPR Modification of the Epigenome

Creating dCas9 protein (dead) which cannot cleave DNA

Then adding gRNA for areas around the CpG Islands

Then rewrite epigenetic markers on both the DNA and the histone proteins

This removes or adds acetyl groups to histones

How can CRISPR technology be used?

Reshape animals, engineer plants, gene therapy, and possibly rewriting humans?

Gene Drives

Self-replicating CRISPR RNA, “selfish genes”

1. A gRNA shepherds the Cas9 cutting enzyme to a specific spot on DNA

2. Cas9 snips the DNA, creating a break in both strands

3. A piece of DNA carrying instructions for making Cas9 and the gRNA matches up with the cut ends

4. The cell heals the cut by inserting the DNA containing instructions for Cas9 and gRNA

Gene drive inheritance

One copy converts gene inherited from the other parent. More than 50% chance of passing it on.

The altered gene is almost always inherited.

What gene drives could do

• Immunize animals that carry humane disease

• Control insect-borne diseases

• Spread pest-specific pesticides and herbicides

• Reduce populations of rodents and other pests

• Control invasive species

  • Aid threatened species

Where gene drives are?

• Mainly hypothetical for wide spread use right now

• Laboratory barriers and techniques

• Need to perfect ensuring the location of gene drives

• Politics