Lecture 24: CRISPR-Cas Immunity
Today�
Remember the Luria-Delbrück Experiment
Inoculate cells onto plate with phage
Small number of colonies will grow
Inoculate these cells onto phage plate
Many more colonies!
Remember the Luria-Delbrück Experiment
When we add the bacterial cells to the phage plate, does that
T1 phage
E. coli
How was did they answer the question?
The two scenarios will have different variances in the number of resistant colonies!
Small variance
Large variance
The two scenarios will have different variances in the number of resistant colonies!
Experimental data
Expected number assuming pre-existing, spontaneous mutations
Long tail!
The two scenarios will have different variances in the number of resistant colonies!
Likely scenario
Small variance
But why not have a way to acquire immunity?
We can do it
Can bacteria?
How can bacteria acquire immunity to phages?
Phage DNA
Bacterial DNA
How can bacteria acquire immunity to phages?
Phage DNA
Bacterial DNA
How can bacteria acquire immunity to phages?
Phage DNA
Bacterial DNA
How can bacteria acquire immunity to phages?
Phage DNA
Bacterial DNA
Cas1-Cas2
How can bacteria acquire immunity to phages?
Phage DNA
Bacterial DNA
Cas1-Cas2
How can bacteria acquire immunity to phages?
Phage DNA
Bacterial DNA
Cas1-Cas2
How can bacteria acquire immunity to phages?
Phage DNA
Bacterial DNA
Cas1-Cas2
The bacterium now has part of the phage DNA in its own genome???
Let’s take a closer look at this locus.
The CRISPR locus
Phage DNA. Called “spacers”.
Spaced out repeat sequences.
Clustered regularly interspersed short palindromic repeats
cas genes (CRISPR associated genes, mostly nucleic acid processing genes like nucleases, etc)
What happens when an encoded phage attempts to infect?
cas genes
Phage sequences
infecting phage genome
Cas proteins
phage-complementary RNAS and “guide” RNAs
What happens when an encoded phage attempts to infect?
cas genes
Phage sequences
infecting phage genome
Cas proteins
phage-complementary RNAS and “guide” RNAs
Cells use multiple types of Cas systems
(+others)
Most famous because it is used for gene editing
Most common
CRISPR systems all search for a short nucleotide sequence called the protospacer-adjacent motif, or PAM, which is short enough to be found in any genome (2-5 bases)
CRISPR systems impart acquired, heritable immunity!
Bacterial genome
CRISPR spacer
CRISPR-Immune cells can grow in the presence of phage
CRISPR immunity
Non-functioning CRISPR
How did researchers discover CRISPR immunity?
Danisco yogurt (now owned by DuPont)
Phages are very bad for yogurt makers
Yogurt factory
Delivery of milk contaminated with phage could ruin the production of all yogurt!
For this reason, dairy scientists were interested in microbes with resistance to phage.
Yogurt scientists hypothesized that CRISPR spacers were associated with immunity
2007 experiment:
Resistant strains acquired new spacers compared to WT
Ratio of plaques produced by strain to that of WT
S. thermophilus CRISPR locus
Removing CRISPR sequences reduces immunity
How widespread is CRISPR immunity?
CRISPR sequences have been found in:
How do CRISPR-Cas complexes find the correct site amid all the DNA in the cell?
Millions of bases!
To answer this question, you must be able to watch CRISPR-Cas9 complexes bind to and move along DNA!
DNA curtains enable measurement of single protein-DNA binding events
fluid flow
Objective
DNA curtains facilitate TIRF imaging
Total internal reflection fluorescence
High refractive index (e.g. glass)
Low refractive index (e.g. water)
Light
Total internal reflection fluorescence
Microscope glass
Aqueous buffer
Fluorophores
A quantum dot-labeled Cas9 enables single-molecule visualization
Cas9
quantum
dot
Fluorescent semiconductor nanoparticle
A quantum dot-labeled Cas9 enables single-molecule visualization
DNA
Cas9
Cas9-RNA complexes bind to DNA sequences determined by the RNA sequence
Cas9 without guide RNA binds DNA, but not in a sequence-targeted way
Cas9
Cas9
+ RNA guide
- RNA guide
Does Cas9-RNA complex bind DNA and search along the DNA or does it diffuse in 3D, hopping on and off the DNA?
Experiment to measure Cas9-RNA searching
Doubly-anchored DNA curtains with no flow
If Cas9-RNA complex binds to DNA and searches along it, you should see labeled complexes moving along DNA.
If instead complexes diffuse in 3D, hopping on and off DNA, you should see transient binding events at specific spots along the DNA.
Cas9 complex hops on and off DNA
Single DNA molecule
CRISPR complex hops on and off DNA, binding transiently, except at target site, where it binds stably
Cas9 complex hops on and off DNA
Time
Off-site binding times:
Why doesn’t the Cas9 complex cut the target DNA at these off sites?
Both target complementarity and a PAM site are required for DNA interrogation and cutting
Experiment:
Modify this sequence
Detect products with a gel
Vary incubation time
Both target complementarity and a PAM site are required for DNA interrogation and cutting
No PAM site results in no binding of Cas9!
Proposed DNA search and cleave model
Note that this also accounts for why the CRISPR-Cas9 complex does not cut the bacterial genome: there is no PAM sequence!
In summary
1
2
3
4
Why has my father heard of CRISPR?
Doubled-stranded DNA
Protein-RNA complex
Cas9
Cas9
DNA with a double strand cut at a sequence determined by RNA-DNA base pairing!!!!
If you can just deliver the CRISPR-Cas9 complex to a cell nucleus, it will cut the genome at a specific location determined by the RNA sequence
Before CRISPR-Cas9, sequence-specific DNA-binding proteins needed to be designed
What have we learned?
A takeaway from this semester
Everything we’ve talked about this semester is happening almost all the time in microbes and communities of microbes
And we’ve barely scratched the surface of what’s going on!!!