CRISPR/CAS9

• What is the first step of CRISPR/CAS9 Mechanisms?

  Immunization
• What is the first step of immunization?

  Acquire the foreign DNA
• How does the cell acquire the foreign DNA?

  The bacteria gets infected with the viral DNA

  If it survives, it stores the foreign DNA in a spacer
• Where is a spacer found?

  Within the CRISPR Locus
• What is the second step of Immunization

  Transcribe the viral DNA into RNA
• True or False: When a new viral DNA is acquired, a new repeat sequence is added into the CRISPR Locus

  True
• When the viral DNA is transcribed into RNA, does it become pre-crRNA or crRNA???

  Pre-crRNA
• What shape do the repeats make once they are transcribed?

  A hairpin loop
• What characteristic do repeats have that allow them to make a hairpin loop once transcribed?

  B/C they are palindromic
• What is the third step of Immunization?

  Process (Cut) the RNA
• True or False: Once the pre-crRNA is cut does it finally become crRNA?

  True
• What does each crRNA contain that is inherited from the pre-crRNA?

  A spacer containing the viral DNA
• What is the fourth step of Immunization?

  Incorporating crRNA into cas9 proteins
• What is the second step of CRISPR/CAS9 Mechanisms?

  Adaptive/Acquired immune Response
• What are the 2 steps that work together in the adaptive/acquired Immune Response

  Targeting by crRNA & Inactivation
• What will happen if the same viral DNA tries to infect the bacteria again?

  crRNA will target the viral DNA by binding to the complementary DNA strands

  Once the crRNA binds it will cause a conformational change in CAS9
• Once the conformational change happens, how does CAS9 finish adaptive Immunity Response?

  The conformational change causes the endonuclease to be activated

  CAS9 will make a double stranded cut and destroy the viral DNA
• What are 2 other components to CRISPR/CAS9??

  gRNA

  PAM
• True or False: crRNA and gRNA are technically the same?

  True
• If crRNA and gRNA are the same then why do they have different names?

  crRNA and gRNA have the same mechanism, the only difference is that gRNA is crRNA replaced with a different RNA sequence that is specific to a DNA region or a gene or interest
• What does PAM stand for?

  Protospacer

  Adjacent

  Motif
• How does PAM work?

  CAS9 recognises and binds to PAM, unwinding that DNA region
• How does CAS9 activate it's endonuclease?

  If the unwound part of the double strand is complementary to the gRNA then it will base pair

  The binding causes a conformational change and the endonuclease is activated
• Where does the cut of the endonuclease occur?

  Upstream of PAM
• Is the break a double stranded break or single stranded?

  It is double stranded but it can result in a blunt end or overhang
• How does gRNA make CRISPR/CAS9 a powerful gene editing tool?

  B/C any RNA sequence can be used in gRNA, meaning, it can target any DNA region and CAS9 will cut it

  It makes everything more precise and less time consuming
• True or False: Once the DNA is cut, it goes thru DNA Repair Mechanism

  True
• What are the 2 DNA Repair Mechanisms?

  HDR

  NHEJ
• What does HDR stand for?

  Homology Directed Repair
• Is HDR similar to deletion or insertion?

  Insertion
• What is the first step of HDR?

  DNA that is cut will get donor Template DNA
• What does Donor DNA consist of?

  Desired Sequence

  DNA homologous (same as) to the blunt end of the cut DNA
• True or False: Naturally, the repair mechanisms of the cell will insert the desired genetic material

  True
• What does NHEJ stand for?

  Non

  Homologous

  End

  Joining
• Is NHEJ similar to Insertion or Deletion?

  Deletion
• Explain the mechanism of NHEJ

  Double stranded breaks in DNA are ligated back together without the help of donor template DNA
• What is the problem with NHEJ?

  If there are missing base pairs in the DNA, NHEJ won't replace them so if any base pairs were deleted in the breakage, they won't be added back in
• Which DNA repair mechanism is more precise?

  HDR
• Which DNA repair Mechanism is faster?

  NHEJ
• What is the problem with NHEJ

  Because you can end up with extra or missing bases, the resulting gene is often unusable or turned off