Lecture_5: CRISPR

 Virus Counter Defenses

• Mechanisms underlying the targeting for Cas1 and Cas2 to viral

genome might be dependent on the linear genome (bacterial
genome is circular)
• Evolve nucleotide changes in virus target sequence, i.e. protospacer
or PAM → mutated PAM → Cas9 cannot recognize if the protospacer
is mutated → Cas9-crRNA cannot cleave it
• Virus encoded suppressor of CRISPR have been identified
⁃ Steps that can be targeted for suppressing the immune
response include all the three stages
• Anti-CRISPR protein have been identified that can inhibit the system
→ also determined the molecular mechanism by which inhibition is
conferred
• Type I-F CRISPR/Cas System
⁃ First anti system was discovered in Type I-F → requires 4
different proteins for targeting (Cys1-4) → interact with CRISPR
RNA → search for regions in phage DNA and can open up the
DNA → Cys1-4 are important for targeting, but for not
cleavage
• Cas3 is the nuclease and is responsible for cleavage
• Cys1,2,4 one each and 6x Cys3 in TypeI-F
• AcrF → AntiCRISPR Type-1 F System
• AcrF1 → 3 protein binds to Cys3 protein → prevents the complex
from attaining alternative confirmation → an example of allosteric
inhibition → allosteric regulation requires complex changes in
protein to enact its effect
• AcrF2 → Interacts with Cys1 and 2 → steric inhibition → physically
blocks crRNA binding to phage DNA
• AcrF3 → inhibits Cas3 recruitment to complex phage DNA
⁃ All three AcrFs attack at targeting stage but in different
processes
• CRISPR-Cas9 Inhibitor
⁃ HNH performs the first cleavage that is base paired to crRNA
→ a conformational change occurs that is essential for second
cut by RuvC → the cleavage is coupled in that one cannot
without the other
• AcrIIC1 → Catalytic sites are specific and therefore the bacteria
cannot overcome the resistance subjected at the catalytic site of
Cas9 by AcrIIC1 → mutations cannot occur at Cas9 because it is a
catalytic site → HNH cleavage inhibition blocks RuvC cleavage
• Paper
 • AcrIIC3 → causes dimerization of Cas9 subunits which in turn inhibits
their ability to cleave the phage DNA → not known whether it is
steric or allosteric

 CRISPR/Cas9 Mammalian Gene Editing

• Type II system was used over Type I-f because it is simpler

• The top of the stem loop separates two RNA in the bacterial system
→ not covalently linked → in this experiment, both the RNAs were
linked covalently
• CRISPR-RNA + tracrRNA → collectively form the guide RNA → needs
to be expressed in the target cell → also requires expression of Cas9
• Sometimes RNAs required for expression are all expressed on a
single viral vector
• Donor DNA is also required
• Gene editing → requires introduction of dsDNA in a particular site in
DNA
• First step in GE is to break the dsDNA → CRISPR/Cas9 uses guide
RNA to precisely cleave a section of DNA
• DNA repair can be NHEJ → a sloppy and inaccurate form of repair →
can results in indels (insertions/deletions) → ligated two broken DNA
strands
• When a donor region that is complementary to a gene is introduced,
the donor DNA will be homologously recombined through HDR
• Donor DNA shares some overlapping regions with the sequence that
is being modified
• For knockout gene → no donor is introduced → makes mistakes
during NHEJ
• Guide sequence is specific to the target → there is a possibility of
off-site indels for sequences similarly to guide RNA

 Antiviral Treatments for HCV

• 71 million who are chronically infected → 400, 000 in 2016

• Extremely variable virus → 6 main genomes with 50 subtypes
• A vaccine against one subtype won’t be functional for multiple virus
• Excellent at evading immune response → NS34A acts on Riplet →
knocks down adaptive immune system due to interconnectivity
• Developing an animal model for vaccine tests is challenging → no
known host for HCV except for humans
• HCV is curable → not a life-long issue → less motivation for
developing vaccines
• Vaccines target the outer glycoprotein on virus → proteins made on
ER → hard to isolate and multiply
 • 250,000 infected in Canada

 HCV Particle and Genome

• Family: Flaviviridae
• Genus: Hepacivirus
• IRES, no poly A tail, and no Cap
• E1 and E2 are hard to produce for vaccine design
• NS3 → catalytic; NS4A is cofactor
• NS5A → multi-purpose protein that facilitates replication of viral
genome and helps in assembly of particles → details still being
worked out
• NS5b → RdRp → RNA dependent RNA polymerase required for
replication of the virus
• Transmission is via blood → mosquitoes are not vector
• Possible transmission through transfusion → strict screening of blood
available to limit this scenario
• Ontario, QC and BC → urban regions with a lot of drug use → hence
a high frequency of HCV infection
• Six major genotypes → G1 important in Canada, States and SA →
actually for much of the world this is the dominant genotype
• Thailand has division of genotypes → no clear distribution pattern
seen for HCV around the world
• North African region most affected by HCV → reasons unknown
• In approximately 15-25% of people who are infected, infection is
clear if they have a well-functioning immune system
• Chronically infected individuals can result in progression to cancer
and cirrhosis of liver (liver scarring)
• Genotype 3 is most difficult to treat