CRISPR & CAR T (finished)

• CRISPR: technique that focuses on methods that could modify DNA sequences in living organisms
• CAR T: involves extracting T cells from a patient and reprogramming them to kill cancer cells
• CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats
• CRISPR and CAR T are methods that have been recently developed to treat patients with diseases that were once unmanageable
• Palendromic repeats from the bacterium are known as spacers
• 5' G A A T T C 3' and 3' C T T A A G 5' is an example of a genomic palindrome
• Jennifer Doudna won the Nobel Prize for the discovery of CRISPR
• CRISPR is an adaptive immune system found in bacteria and archaea
• Cas proteins are what store pieces of phage DNA as a memory of infection

  • Memories are used to guide and find matching phage genomes and destroy them to stop subsequent infection
• CAS 1 and CAS 2 excise DNA
• CrRNA detects unwanted DNA and RNA and is used to remove unwanted DNA and RNA from the cell
• PAM sequence: short sequences of DNA located following target DNA that is recognized by Cas9

  • A marker or used as a guide
  • Repeats in CRISPR array don't contain PAM
• In prokaryotes, CRISPR is used to protect bacteria from phage infections through memory and immune system
• sgRNA stands for single guide RNA

  • Used in lab
• In eukaryotic cells, CRISPR and Cas9 are used for DNA/gene editing
• CRISPR in nature (prokaryotic)


  1. New viruses are integrated into CRISPR array
  2. CRISPR array is transcribed and processed into crRNA (CRISPR RNAs)
  3. crRNA binds to CRISPR effector complex to initiate recognition and degradation of foreign DNA or RNA
• Ribonucleoprotein (RNP) complex is made up of guide RNA (gRNA) and SpCAS9 nuclease

  • PAM is required for gRNA to bind to a target sequence
  • SpCAS9 breaks double strand in DNA
• Inactive Cas9 (dead Cas9) binds to target sequence using sgRNA and blocks RNA polymerase

  • Recruits transcriptional activators
• Genome editing:

  1. Identify the gene of interest
  2. Cut the gene of interest
  3. Splice in the gene of interest
• Base editing: single base modifications that can be used to correct mistakes in DNA
• Gene regulation:

  1. DNA is transcribed into mRNA
  2. mRNA is translated into a polypeptide or mRNA is blocked from inactive Cas9
• CRISPR in lab (in eukaryotes)

  1. Design a guide RNA (gRNA) that's specific to target gene
  2. Deliver gRNA and Cas9 into nucleus for binding to target DNA sequence
  3. Induce double strand breakage
  4. The cell's natural DNA repair mechanisms will introduce desired edits
• 2 types of DNA double strand break repair


  1. End joining
  2. Homology-directed repair
• CRISPR in Sickle Cell Disease: can be treated by editing hematopoietic stem or progenitor cells


  • Remove and edit targt cells in the lab then return cells to host
  • Directly deliver CRISPR-Cas editing tools to the affected tissue (in vivo)
• Repressor proteins are used to turn off gene transcription. Its target is Cas9
• Activator proteins are used to increase gene transcription. Its target is Cas9
• First CRISPR therapy: Casgevy was approved to treat sickle cell disease in December 2023
• CAR T stands for chimeric antigen receptor T cell therapy
• First generation of CAR T was not very successful, but the most recent generation was successful
• First CAR T therapy: 2017, CAR T cells from a patient’s own T cells were used to treat a patient with leukaemia
• CAR T are not good at treating solid tumors; rather they target leukemia and lymphoma
• Steps of CAR T Therapy:

  1. Remove blood from patient to obtain T cells
  2. Make CAR T cells in lab
  3. Amplify and make more CAR T cells
  4. Infuse CAR T cells into patient
  5. CAR T cells bind to cancer and kill them
• The CAR molecule is a potential target for gene editing to increase binding efficacy through CAR T therapy
• CAR molecule is generally derived form monoclonal antibody
• CD19 is a marker for B cells and is expressed on cell surfaces and involved in many signaling pathways
• SLE have intense B cell activation, which leads to the production of autoantibodies
• Cytokine Release Syndrome (CRS): side effect of CAR T therapy
• Anakinra targets IL-1 to inhibit neurotox
• Lenzilumab targets GM-CSF to inhibit neurotox
• Metyrosine inhibit sactivation loop in macrophages