Welcome to Dr. Kate Kraus Brilakis' Learning Portal

the CRISPR system in  bacteria functions like our immunomemory

 Repeats are 30 bp segments of DNA that all have the same sequence. 
​Each repeat is palindromic.
a stretch of bases is followed by a stretch of the the complementary bases of the same sequence in reverse order. like RACECAR.
this allows the repeats to fold into a hairpin like structure once they’re transcribed into RNA.

in the human immune system,
when a virus infects us, we produce
antibodies to that specific virus = immunomemory.
When the same virus infects us again, these antibodies recognize the pathogen and can eliminate it. 

can anyone guess the role of the PAM?

back to crispr...

teeny bit more important info...
there's a sequence of DNA called the
 PAM (Protospacer Adjacent Motif).
this sequence is found downstream from where the viral DNA is cleaved.

The CAS enzyme of the bacteria hunts for the PAM and that's where the enzyme cuts the DNA to make the spacer that will be inserted into the bacterial genome. 

an example of a PAM, which is different for every bacterial species, is the sequence 5' NGG 3' that is the PAM for Streptococcus pyogene.

<-- this is an enzyme

logic circuits

ethical considerations
​CRISPR genomic editing

 in research:
1. illustrate gene function:
   "knock out" or modify specific genes in cells or model organisms (mice) to
     understand the function of the gene.

2. develop therapies:
     identify vulnerabilities in cancer cells to hopefully lead to targeted therapies 

3. study mechanisms of disease:
    create modified cell lines that model diseases permitting the study of
    a disease and test potential treatments.

1987: The initial observation of unusual, repetitive DNA sequences in E coli 
            by Yoshizumi Ishino and his team in Japan.

1993-2005: Francisco Mojica in Spain recognized common CRISPR features across                          different bacterial species. He postulates CRISPR functions as an                                    adaptive immune system after discovering that CRISPR sequences                                  matched viral snippets.

early 2000s: CRISPR sequences linked with associated Cas proteins 

2012: Emmanuelle Charpentier and Jennifer Doudna (and teams) demonstrate that
           the CRISPR-Cas9 system could be engineered as a programmable gene-editing
           tool. They were awarded the 2020Nobel Prize in Chemistry

sooo....DNA sequence determines RNA sequence.
​RNA sequence determines amino acid sequence.
Amino acid sequence determines protein's shape.
Protein's shape determines protein's function

what about the PAM?

 all enzymes are proteins.  enzymes facilitate reactions. Nuclease enzymes cut DNA. 

virus vs bacteria

how has CRISPR  been harnessed?

CRISPR 
permits the precise modification of DNA sequences within living organisms. 
CRISPR was originally discovered in 1987 as a microbial "immune system" used to recognize and eliminate specific viral pathogens.

 in agriculture:

7. CRISPR is being used to develop crops that are resistant to diseases, pests, 
    drought and extreme temperatures.

8. CRISPR is being used to increase crop yields, grain size, and nutritional                content in crops like rice, wheat, and soybeans.

9. CRISPR is being used to create crops that can capture more carbon from the       atmosphere and store it in their roots to help mitigate climate change. 

the rapid development of CRISPR technology requires we participate in ethical debates. public engagement and education must be encouraged to ensure responsible development and application.

​CRISPR and sickle cell

applications:

the memory of the phage infection is now in the bacterium's DNA.
this DNA memory will be used as a template to produce a lot of copies of RNA with the same memory sequence
= crispr RNA.
 Cas proteins bind with crRNA forming a
surveillance complex
which roams the bacterium,
​hunting for matching phage DNA.

below is a distilled explanation of how the CRISPR system is used for gene editing

RNA v DNA

 in medicine:
4. treating genetic disease:
    CRISPR is being used in clinical trials to treat inherited blood disorders               like sickle cell anemia and beta-thalassemia (more below). the faulty genes in an patient are edited/replaced by correct DNA coding.

5. cancer immunotherapy:
    CRISPR is being used to alter immune cells/T-cells to target cancer cells

6. organ transplantation:
     CRISPR is being used to genetically modify pig organs (69 genes modified)         so their compatible for human transplantation. 

Clustered Regularly Interspaced Short Palindromic Repeats

the acronym CRISPR =
 clustered regularly interspaced short palindromic repeats
refers to sections of DNA identified in bacterial genomes. CRISPR is part of an array
​that contains 
two components:
 repeats & spacers.

silencing the gene that codes for a protein that acts to repress the expression of the
​fetal hemoglobin gene =

turns on fetal hemoglobin gene

let's first recap some info...

replace the mutation with the "correct" nucleotide ​  

step 2: reinfection
the invading phage is destroyed by a process 
called interference.

when a phage injects its DNA into a bacterial cell, surveillance complexes scout the phage’s genome for a sequence that matches it's crRNA.
*crRNA is a complement of the original phage DNA.
the surveillance complex unzips the phage DNA and checks to see if the crRNA can pair up with it. If the whole spacer can base-pair, it means the surveillance complex has found the target it’s been searching for.
it cuts the phage DNA to destroy it.
​If the surveillance complex searches through all the DNA in the cell but doesn’t find a match, it doesn’t make any cuts.

bacteriophage
= virus that targets bacteria

step 1: acquisition:
making the memory

phage infects a bacterium.
two Cas proteins (Cas1 and Cas2) work together to cut out pieces of the phage DNA and inserts it into one end of the CRISPR array as a new spacer.
A new repeat is also added so all spacers are always flanked by repeats on each side. A memory of the phage is stored. 
Think vaccination!

cancer targeting via CAR-T and CAR-NK

crispr

human germline editing
CRISPR can be used to modify the human germline (sperm, eggs, and embryos), meaning these changes would be passed down to future generations. This raises concerns about unforeseen consequences and the potential for unintended mutations to be introduced into the gene pool. 

unintended health effects
 CRISPR could have unintended effects on other genes,
leading to unintentional medical issues 

designer babies
will CRISPR be used for non-therapeutic purposes?
can traits such as intelligence or athleticism be cultivated?
could there be a future where genetic enhancements are available to the wealthy?

eugenics
could CRISPR be used to select for certain "favorable" traits leading to discrimination and the marginalization of individuals with "unfavorable" genetic characteristics. 

access/equity
CRISPR could exacerbate existing health and social inequalities.

safety/oversight

off-target effects may produce unintended consequences

informed consent
 understanding the potential risks vs benefits 

impact on human diversity
could the widespread use of CRISPR to eliminate genetic diseases lead to a decrease in human diversity?

ecosystem impact
 unintended consequences on ecosystems (plant editing) needs to be carefully considered

when a virus infects a bacterial cell, CRISPR also creates immunomemory.
the bacterium takes a piece of the virus’s genome and inserts this viral DNA into its own genome. The bacterium uses this sequence of the viral DNA to create a memory surveillance system that can
disable any virus of the same species  that might reinfect the bacterium.

Spacers are found between the repeats. Each spacer is a small segment of unique phage DNA...the “molecular memory” of a previous infecting virus. These spacers are passed down through generations of bacteria so the individual bacterium doesn’t have to have been infected in order to be immune to a phage. 

alzheimers disease

​explain the CRISPR acronym...

the sequence of the
amino acids (peptides)
​ determines how the amino acids  interact with one another along the long polypeptide chain. this interaction determines how the protein folds which determines the protein's shape/structure which determines the function of the protein. 

"orphan" diseases

  two routes for a cure

what DOES happen now??

epigenomic editing

let's go step by step...

purpose of RNA v DNA

the palindromes (repetitive DNA sequences) of CRISPR were first observed in 1987 but not understood to be part of a bacterial defense mechanism.