BIMM 100 Final Exam (not cumulative)

covers LE 14-18, week 9-10 DI slides, final exam review slides (+podcast, watch if have time), problem sets 7+extra, MB questions from BioConnect final exam review slides

def. epigenetics (← give 2 def.)

• describe (2)

• can be affected by __ and the __

name the 3 predominant forms of epigenetics

changes in the genome (gene expression & genome) that don’t change the gene/DNA sequence

• specific/some epigenetic modifications can be passed down from parent to daughter cell & can affect more than one generation

• changes are reversible

• can be affected by diet and the environment

how your behaviors and environment can cause changes that affect how your genes work (← other definition)

__

epigenetics predominantly appears as:

• histone modifications

• non-coding RNAs

• DNA methylation

(^ histone mod. talked about in previous midterm LEs, other 2 are new)

most RNAs in eukaryotic cells are __, specifically what 2 kinds?

in terms of non-coding RNAs, what are the 2 general categories of ncRNAs?

• function of each category ncRNA (1) (2)

there are many types of long non-coding & small non-coding RNAs, where __ is a type of small non-coding RNAs

• what deter. if small or long RNAs? aka give length of each

regulatory ncRNAs form different structures, ex: __ __ / they don’t exist as a straight line

most RNAs in eukaryotic cells are non-coding RNAs (ncRNAs), specifically what long non-coding RNAs (lncRNAs) & micro RNAs (miRNAs)

__

2 general categories of ncRNAs:

• housekeeping ncRNAs — regulate translation

• regulatory ncRNAs — regulate gene expression & mRNA stability

__

there are many types of long non-coding & small non-coding RNAs, where miRNAs is a type of small non-coding RNAs

• small RNA if 20-25 nucleotides

• long RNAs if >200 nucleotides

regulatory ncRNAs form different structures, ex: hairpin structure / they don’t exist as a straight line

describe micro RNAs / miRNAs (3 ← incl. function/result)← type of small non-coding RNA

t/f: miRNAs are processed in the nucleus & cytoplasm, and then in the cytoplasm, miRNA will interact w/ its target mRNA to determine the outcome

• what is it called when miRNA interacts with its mRNA target?

  • name 2 ways this can occur & what part(s) of the mRNA target is involved in each

  • which is exact/precise vs imprecise pairing?

• pairing b/w a miRNA and its mRNA target depends on the __ __ (← is how long?)

miRNAs usually __ mRNAs from undergoing translation, so there is __ protein expressed

• are encoded in the genome

• are small RNAs of 20-25 nucleotides long

• cause degradation (through cleavage) to inhibit translation of the mRNA target

__

true: miRNAs are processed in the nucleus & cytoplasm, and then in the cytoplasm, miRNA will interact w/ its target mRNA to determine the outcome

• miRNA interacts with its mRNA target via standard base-pairing by:

  • near perfect/perfect complementarity in the coding region OR UTR (of the target mRNA) (that does exact pairing)

  • short complementarity in the 3’ UTR (that does imprecise pairing)

• pairing b/w a miRNA and its mRNA target depends on the seed sequence ← ~6 nucleotides

_

miRNAs usually inhibit mRNAs from undergoing translation (so less protein is expression)

for exact pairing / near perfect complementarity in coding region or UTR

what is the outcome?

• this will affect the __ of an organism ← explain w/ ex. of HOXB8 gene and miR-196 (general statement)

  • what happens to HOXB8 when miR-196 is overexpressed?

  • what happens to HOXB8 when miR-196 is reduced?

exact pairing extends beyond the __ sequence

____

for imprecise pairing / short complementarity in 3’ UTR

outcome (2)

mRNA degradation via target mRNA cleavage

• this will affect the traits of an organism

  • miR-196 cleaves the HOXB8 mRNA, which limits the expression of the HOXB8 protein

  • when miR-196 is overexpressed → cleaved & degraded target mRNA → HOXB8 decreases → so shorter fish (than wt)

  • when miR-196 is reduced → increased mRNA → HOXB8 increases → so longer fish (than wt)

exact pairing extends beyond the seed sequence

____

for imprecise pairing

• inhibit translation

  • (b/c reduced ribosomal activity)

• destabilization b/c degradation via deadenylation (remove 3’ polyA tail)

  • (where mRNA will be degraded, so there’s less mRNA to translate)

____________________

^^ shows how the outcomes of both miRNA-mRNA pairings result in less protein (due to inhibition of certain processes)

describe lncRNAs (3 ← incl. function/result)

• for the 2 lncRNA functions/results above, describe the 2 effects/functions for each

• are encoded in the genome

• long RNAs w/ >200 nucleotides

• many activities, including reg. gene expression by/via affecting chromatin & post-transcriptional reg.

reg. of gene expression:

• promote open chromatin

• promote closed chromatin

post-transcriptional reg.:

• direct reg. of mRNA stability

• indirect reg. of mRNA stability by involving miRNAs

for post-transcriptional lncRNA to reg. mRNAs

lncRNAs can directly stabilize mRNAs by binding to __

lncRNAs can indirectly stabilize mRNAs by involving __, where these lncRNAs are called __ (← stands for)

• how does this work?

lncRNAs can directly stabilize mRNAs by binding to mRNA

lncRNAs can indirectly stabilize mRNAs by involving miRNAs, where these lncRNAs are called ceRNAs (← competing endogenous RNAs)

• ceRNAs have complementary seq. to the miRNAs, so they bind together to move miRNA away from the target mRNA (SO less miRNA will cleave and degrade the target mRNA)

for reg. gene expression of lncRNA by affecting chromatin

some lncRNAs can promote __ & some can facilitate/cause __ chromatin

this is seen w/ X-inactivation (← def.)

• state the 3 steps

  • though keep in mind that methylation does NOT determine if chromatin is closed (hetero-) or open (eu-). It is just that most condensed chromatin (heterochromatin) has __ histone proteins

^ Xist is a __

^ spec. how does Xist work?

_____

review:

3 main histone tail modifications

transition b/w chromatin states is reg. by (1), where lncRNA tends to turn __ chromatin into __ chromatin

some lncRNAs can promote open chromatin & some can cause closed chromatin

seen w/ X-inactivation — ONE X chromosome is inactivated by a lncRNA in females, while the other X chr. is active

• BOTH X chr. are active, so genes are expressed. There is also acetylated histone tails, so the chromatin is open.

• SAFA (scaffold attachment factor A) recruits Xist, where Xist will inactivate ONE X chr. in female cells; AND Xist will recruit HDAC (histone deacetylase), which deacetylates histone tails

• Xist also recruits a complex containing histone methyltransferase, which methylates histone tails → SO chromatin is condensed

  • though keep in mind that methylation does NOT determine if chromatin is closed (hetero-) or open (eu-). It is just that most condensed chromatin (heterochromatin) has methylated histone proteins

^ Xist is a lncRNA (lncRNA that promotes closed chromatin in reg. of gene expression)

^ spec. Xist that inactivates 1 X chr. in female cells will spec. inactivate genes that are close to the Xist gene, b/c SAFA binds in many places & the recruited HDAC will cause chromatin compaction/condensed/closed that locally spreads where SAFAs exist

____________

review:

3 main histone tail modifications: acetylation, methylation, phosphorylation

transition b/w chromatin states is reg. by histone tail modifications, where lncRNA tends to turn active/open chromatin into inactive/condensed/closed chromatin

def. coding vs. noncoding strand

lncRNAs are encoded in the genome in many places, so there are many types of lncRNAs

• name 7 types (& describe 5 of them)

coding strand (sense strand) — DNA strand whose sequences matches the transcribed mRNA

• (mRNA and DNA coding/sense strand have the same 5’ and 3’ end directions)

noncoding strand (antisense strand) — DNA strand where lncRNA is transcribed from / complementary to coding strand

• (mRNA and DNA noncoding/antisense strand have the opposite 5’ and 3’ end directions)

__

intergenic

• b/w 2 (coding) genes

bidirectional

• made from a bidirectional promoter, so can transcribe RNA in either direction (direction of exon OR direction of lncRNA)

antisense

• antisense lncRNA is on the antisense strand aka complementary to the coding strand that produces mRNA (aka comp. to strand that makes mRNA)

sense-overlapping

• is on the sense/coding strand, BUT the lncRNA’s TSS is different from the coding gene’s TSS

intronic

• is w/in an intron, but intronic lncRNAs can’t be spliced out & has to be independently translated from mRNA

enhancer lncRNA (lncRNA in enhancer)

promoter lncRNA (lncRNA in promoter)

for epigenetics: DNA methylation

the most common type of DNA methylation is (1)

• is it mutagenic? explain

cytosine (and DNA in general) is methylated by __ (← stands for?) to form this common DNA methylation product

while DEmethylation of DNA involves the enzyme __ (← stands for?)

____

review:

draw the 4 types of nucleotide bases & state if purine/pyrimidine

the most common type of DNA methylation is 5-methylcytosine

• not mutagenic b/c doesn’t disrupt H bonding b/w C-G base pairs, so no change in DNA sequence/ not mutagenic

cytosine (and DNA in general) is methylated by DNMT (DNA methyltransferase) to form this common DNA methylation product: 5-methylcytosine

while DEmethylation of DNA involves the enzyme TET (ten eleven translocation enzyme)

for epigenetics: DNA methylation

2 parts of the genome that are usually methylated / have methylated CpG islands

1 part of the genome that is usually NOT methylated ← why?

• def. islands

in general, DNA methylation is usually ass. w/ __ gene expression, but is sometimes ass. w/ __ gene expression

_

loss of transposon methylation can cause (2)

____

review:

t/f: more than half of human genome is made of transposable elements

usually methylated / have methylated CpG islands:

• transposons/transposable elements

• transcription unit of genes

usually NOT methylated:

• CpG island promoters

  • b/c methylation of CpG island promoters will silence gene expression

• islands — UNmethylated CpG sequences

in general, DNA methylation is usually ass. w/ reduced gene expression, but is sometimes ass. w/ increased gene expression

_

loss of transposon methylation can cause

• transposon activity that destabilizes the genome

• mis-reg. of nearby gene expression

(review: true)

for epigenetics: DNA methylation

for loss of transposon methylation that can cause misreg. of gene expression, which affects traits:

what are the 2 phenotypes of mice?

def. Agouti gene

a __ called __ is inserted upstream of the Agouti gene & present in __ mice

____

review:

3 differences b/w retrotransposon & DNA transposon

what does upstream mean?

yellow fur, obese & brown fur, normal weight

Agouti gene — produces a protein that regulates fur color & body weight

a retrotransposon called IAP is inserted upstream of the Agouti gene & is present in some mice

____

review:

3 differences b/w retrotransposon & DNA transposon:

• retro- do copy+paste, DNA- does cut+paste

• retro- are transcribed into an RNA inter. via RNA pol.

• the RNA inter. in retro- is then converted into DNA via RT (reverse transcriptase)

upstream — sequence that’s 5’ of the promoter

cont. Agouti gene

insertion of IAP causes Agouti to be __, resulting in what pheno?

what happens when the inserted IAP is methylated? why? what pheno?

^ reworded as high IAP methylation vs. low IAP methylation causes what?

__

why does IAP methylation vary to get a spectrum of DNA methylation?

• aka cytosine methylation patterns are affected by (3)

  • describe 1 of them

• note: DNA methylation is enzyme-mediated by __ that gets __ __ from our __, so it is not spontaneous

insertion of IAP causes Agouti to be overexpressed, resulting in yellow fur & obese

inserted IAP is methylated / methylation of IAP → Agouti is NOT overexpressed aka get normal Agouti expression (b/c methylation of IAP suppresses IAP from increasing Agouti expression) → brown fur & normal weight

^ high IAP methylation → Agouti is NOT overexpressed — brown fur, normal weight

^ low IAP methylation → Agouti is overexpressed — yellow fur, obese

__

cytosine methylation patterns are affected by experience, chemicals, and diet

• folate & folic acid in food affects the amount of methyl groups consumed, which then affects DNA methylation (for epigenetics)

• note: DNA methylation is enzyme-mediated by DNMT that gets methyl groups from our diet, so it is not spontaneous

cont. Agouti gene

if a pregnant mouse has IAP inserted upstream of Agouti & is fed food low in methyl groups, what happens to the retrotransposon IAP? pheno?

• pheno of offspring?

but if fed food supplemented in methyl group donors, then what happens to IAP? Agouti expression? pheno?

__

^^ overall, the foods you eat affect your __ & your __ cells, which would affect the next gen./your offspring

…

which is the wildtype pheno?

__

diet of the pregnant mouse affects __ generations (aka affects __ and __)

__

summary:

epigenetic diet provides building blocks for a healthy genome, where __ in diet provides __ __, which is used by different __ to do __ & __ __

t/f: a good example of multi-generation epigenetic effect is the Dutch Hunger Winter famine

t/f: fathers’ diets also have significant epigenetics effects on their offspring

(poor diet:)

IAP is weakly or NOT methylated → yellow fur & obese/poor health

• (b/c low methyl groups, which cause overexpression of Agouti, so yellow fur and obese)

offspring mainly look like mother w/ yellow fur & obese/poor health, but can still have offspring w/ brown fur & normal weight/good health

__

(healthy diet:)

IAP is methylated → Agouti gene is not overexpressed aka normal → brown fur & normal weight/good health

offspring mainly look like mother w/ brown fur & normal weight/good health, but can still have offspring w/ yellow fur & obese/poor health

__

^^ overall, the foods you eat affect your genome & your germ cells, which would affect the next gen./your offspring

…

wt pheno. is brown fur & normal weight/in good health

__

diet of the pregnant mouse affects TWO generations (aka affects children and grandchildren)

• (b/c F1 fetus growing in the pregnant mouse already has F2 germ cells)

__

summary:

epigenetic diet provides building blocks for a healthy genome, where folate in diet provides methyl groups, which is used by different enzymes to do histone &DNA methylation

_

true (multi-generation epigenetic effect is famine)

true (fathers’ diets also have significant epigenetics effects on their offspring)

overview:

want to keep transposons methylated to prevent __ the genome & __ of gene expression

_______________________________

gene expression is regulated by a combination of (2) ← aka crosstalk

• where some histone tail mod. are enriched/located in __ DNA, while other histone mod. are enriched/located in __ DNA

  • SO we can’t assume that methylation of histone proteins will correlate with __ or __ in gene expression, specifically can result in __ or __ enzymes to modify DNA + histone proteins

explain pic

____

(in this ex. w/ MeCP2:)

methylation of DNA by __ (which is a “__”) will be recognized by “__” called __ (stands for?), specifically __

describe the further process of this (3)

→ SO DNA methylation is typically ass. w/ __ gene expression

^^ t/f: methylation of genomic DNA / DNA methylation will recruit chromatin remodelers, including __

…

• what’s the effect of deacetylating histones?

want to keep transposons methylated to prevent destabilizing the genome & misregulation of gene expression

_______________________________

gene expression is regulated by a combination of DNA methylation & histone tail modifications

• where some histone tail mod. are enriched/located in methylated DNA, while other histone mod. are enriched/located in unmethylated DNA

  • SO we can’t assume that methylation of histone proteins will correlate with increase or decrease in gene expression, specifically can result in blocking or recruiting enzymes to modify DNA + histone proteins

…

(pic)

• DNA methylation & histone mod. (H3K36me2) will block the enzyme (PRC2 from methylating histones)

• histone mod. (H3K36me2) will recruit enzyme (DNMT3A), which methylates DNA

____

(in this ex. w/ MeCP2:)

methylation of DNA by DNMT (which is a “writer”) will be recognized by “readers” called MBD (methyl-binding proteins), specifically MeCP2

• (in methylated DNA) MeCP2 recognizes & binds to methylated CpGs

• MeCP2 recruits HDAC (histone deacetylase)

• deacetylation of histone tail will promote chromatin formation aka chromatin compaction/condensation, which blocks gene expression

→ SO DNA methylation is typically ass. w/ reduced gene expression

^^ true: methylation of genomic DNA / DNA methylation will recruit chromatin remodelers, including HDACs

…

deacetylating histones via HDACs will restore the “+” charge on lysine, which strengthens the electrostatic attraction b/w histone & DNA → promotes wrapping DNA around histones to get chromatin compaction/condensation

for DNA methylation

what happens to DNA methyl. as organisms age? (3, where 1 of them has 2)

__

def. epigenetic clocks (another name?)

• aka is the robust correlation b/w changes in __ __ & age

what changes in DNA methylation (& resulting changed in gene expression) occur in what specific parts of a genome (comparing child & older adult)? (← 2)

generally, DNA methylation decreases w/ age in humans

• decreased DNA methylation (will change how DNA is packaged)

• altered histone modifications, where:

  • increase of activating histone mod.

  • decrease of repressive histone mod.

  • (^ both for more open chromatin, so can do gene expression ← shown in below point too)

• global reduction in heterochromatin (closed chromatin)

__

epigenetic clocks / Horvath’s clock — align epigenetic age w/ chronological age (aka predict chronological age w/ DNA methylation of epigenetic age)

• aka is the robust correlation b/w changes in DNA methylation & age

in older person:

• decreased methylation of “non-CpG island”/methylated promoters → increased gene expression

• increased methylation of “CpG island”/unmethylated promoters → decreased gene expression

• decreased methylation of transposons → increased transposon expression

(remember that methylation of CpGs in promoter or transposon will decrease or silence gene or transposon expression)

what mutation alter the methylation of a gene, which affects that gene’s expression? (1 ← incl. how & the result)

• describe the gene (1)

does this mutation affect protein expression? explain

mutation: increased CGG repeat expansions that increase DNA methylation to get methylated repeats → FMR1 gene silencing

• FMR1 / FRAXA gene (Fragile X gene) has a CGG repeat in the 5’ UTR of the mRNA

(^^ mutation can be called FXS)

doesn’t affect protein expression

• b/c the affected CGG repeats are in the 5’ UTR, which is a non-coding region for proteins

originally, gene editing relied on __ __, such as __ __

limitation of gene editing (1 ← explain)

__

2 types of mech. to repair double-stranded break/DSB in DNA

• which is precise vs. error-prone

researchers will make a researcher-designed __ __ to pair with the target site in the genome

_

briefly describe steps preceding homologous recombination (2)

• the introduced homologous sequence can either come from __ __ or __ __

drawback of homologous recombination (1)

so, what is done instead?

…

—→ so now that you used an endonuclease to create a DSB / nuclease-induced DSB, it can undergo 2 different DNA repair pathways: (2)

originally, gene editing relied on homology-directed repair (HDR), such as homologous recombination

limitation of gene editing:

• genomes are constantly changing b/c get mutations due to replication, aging, and the environment & b/c have many transposons that can affect the genome

__

repair double-stranded break/DSB in DNA via HDR/homology-directed repair (precise) or NHEJ/non-homologous end-joining (error-prone)

researchers will make a researcher-designed donor template to pair with the target site in the genome

__

lab-created allele containing the researcher-designed donor template in introduced into the cell → pair w/ target site in genome → homologous recombination can occur

• either the donor template OR a sister chromatid can be introduced that contains homologous sequences

drawback:

• frequency of homologous recomb. in many organisms is low, so can’t rely on this method to edit a genome

SO instead, use an endonuclease to create site-specific DSBs that can increase frequency of DNA repair pathway (at specific site in the genome) SO can do gene editing

…

—→ so now that you used an endonuclease to create a DSB / nuclease-induced DSB, it can undergo 2 different DNA repair pathways: HDR via homologous recombination & NHEJ

---

(So the purpose of inducing a DSB is not because DSBs don't exist, but because we need a DSB at the precise target site to strongly stimulate repair and editing there

Without a targeted DSB, the cell has little reason to incorporate your donor DNA at that particular locus, so homologous recombination remains very inefficient.

Think of it this way:

• Natural DSBs = random potholes scattered throughout the genome.

• CRISPR-induced DSB = deliberately digging a hole at the exact address you want to renovate.)

for DNA repair pathway: HDR

process of HDR: homologous recombination requires (1 ← could be 1 or 1)

what is name of a predominant HDR pathway?

• steps of this pathway (6)

• this HDR pathway does NOT involve __, so doesn’t form __ __

draw out this specific HDR pathway

process of HDR: homologous recombination requires homologous sequence either from homologous allele on sister chromatid OR researcher-designed donor template containing homologous sequence

predominant HDR pathway: SDSA (synthesis-dependent strand annealing)

• endonuclease creates a DSB in DNA

• 5’→ 3’ exonuclease will make 3’ overhangs

• ONE 3’ overhang finds & anneals to the homologous sequence (in repair template)

• that 3’ overhang is extended by DNA pol.

• this newly synthesized strand will reanneal to the broken end

• the gap in the complementary strand is filled in via DNA pol. that uses the newly synthesized strand as the template

^ the DNA in SDSA pathway does NOT crossover, so doesn’t form Holliday junctions

---

(the donor template is in blue, the repair template provided is in red)

for DNA pathway: HDR, spec. SDSA

SDSA is directional, what does this mean?

• so if there is mutation A upstream of the 3’ end & mutation B downstream of the 3’ end when it anneals, then whifch mut. is included?

(^ when the goal is to introduce a change to the endogenous DNA sequence)

SDSA is directional, meaning that the 3’ overhang that initiates repair will determine which sequences in the repair template are included

in this “ “ case, mut. B is incorporated b/c it’s downstream (and the 3’ end that anneals will extend via DNA pol in the 5’ → 3’ direction to include that mutation B), and mut. A is not incorporated b/c is upstream of the 3’ end repairing the DNA break

mut. A and B are incorporated, not mut. C and D

• the starred 3’ end is comp. to the upper blue strand

• b/c DNA synthesis is 5'-to-3' (new nucleotides added to the 3' end), and thus

  DNA will be synthesized to the "left" of the double-strand break. The newly

  synthesized DNA will only include mutations in the template that correspond

  to the "left" of the double-strand break.

for DNA repair pathway: NHEJ

describe NHEJ

• does NOT rely on (2)

• result is called __, where that will be variable in length

__________

summary of both DNA repair pathways:

• researchers can control (2)

• while the cell ultimately deter. (1)

also, which repair pathways are done in dividing and/or non-dividing cells?

broken DNA ends are fused together

• does NOT rely on homologous sequences or donor templates

result is called indels, where that will be variable in length

__________

summary of both DNA repair pathways:

• researchers can control where DSB occurs & if donor template is given or not

• while the cell ultimately deter. which DNA repair pathway is used (to repair the break)

HDR via SDSA in dividing cells

NHEJ in dividing & nondividing cells

what are the 2 commonly used DNA-binding domains used in making a DSB?

problem w/ using either of these 2 DNA-binding domains

^^ how they work: these DNA-binding domains ZFNs and TALENs are made of __ __ (← name 2) that are fused to an __ (← name)

• where… (aka how do they form DSBs)

t/f: both parts of the DNA-binding domain have to be in pairs to create the DSB

• zinc finger nucleases (ZFNs)

• TAL effector nucleases (TALENs)

problem: ZFNs and TALENs are hard to produce

^^ how they work: these DNA-binding domains ZFNs and TALENs are made of transcription factors (TALEs, ZFs) that are fused to an endonuclease (Fok1)

• where ZFNs and TALENs are modified to recognize specific DNA sequences & direct the Fok1 endonuclease pairs to cleave DNA where these DNA-binding domain proteins/TFs pairs (TALE pairs, ZF pairs) bind at

_

true

---

aka

ZFNs and TALENs each have to be used in pairs, and Fok1 is the endonuclease used to cut the DNA when in pairs (Fok1 pairs) at specific places on the genome that are directed by ZFN pairs or TALEN pairs → create a DSB

about a decade ago, we started using the gene editing machinery called CRISPR-Cas9:

what are the 2 parts of CRISPR-Cas9? def. each

• describe (3 each)

__

diff. b/w TALENs and ZFNs vs. CRISPR-Cas9 (in terms of knowing where the genome target is & how it is cleaved to make DSBs)

how do they all relate to the DNA repair mechanisms/pathways?

what are the 2 nuclease domains in Cas9?

Cas9 — an endonuclease that has 2 endonuclease domains (where each endonuc. domain is like a catalytic regions)

• the 2 endonuc. domains create a/one DSB

• ONE endonuclease domain can be mutated to become a nickase / Cas9 nickase

• Cas9 doesn’t bind to DNA, but is instead recruited by guide RNA to attach to the DNA sequence

guide RNA (gRNA) — guides Cas9 to the target genome

• guide RNA is short: 20 nucleotides

• is highly specific, where guide RNA binds to the target genome via standard base-pairing w/ H-bonds

____

• ZFNs and TALENs recognize where to cleave genomic DNA via sequence-specific DNA-binding domains; cleave via Fok1 endonuclease pairs → DSB

• Cas9 relies on guide RNA to where the target genome is, b/c Cas9 does NOT have DNA-sequence specificity; cleave via 2 nuclease domains of Cas9 that cleave both strands simultaneously → DSB

Cas9, ZFNs, and TALENs create DSBs that are repaired by DNA repair pathways of HDR via SDSA or NHEJ

…

Cas9’s 2 nuclease domains are RuvC1 & HNH

what is required for the CRISPR-Cas9 system? (~4/5)

Cas9 2 key functions (aka Cas9 has activity and activity) ← just name

describe how the CRISPR-Cas9 gene editing works (via the 2 key functions above)

• Cas9

• gRNA

• target DNA sequence

• PAM site

• optional is the (researcher-designed) donor template

__

Cas9 has:

• helicase activity

• endonuclease activity

__

• Cas9 helicase activity: Cas9 denatures the target DNA helix, so gRNA can form standard base-pairing H bonds w/ the target sequence

• Cas9 endonuclease activity:

  • Cas9 complex scans for the PAM site (is 5’-NGG-3’) & to see if the target sequence matches/is ~same as the gRNA

  • Cas9 complex cleaves 3 nucleotides upstream (5’) from PAM

& indicate translational start site w/ an arrow

__

Which DNA repair mechanism would result in a mutation if a donor template is not used? What type of mutation could be introduced & where in the given DNA?

• b/c the mutation is at this specific place in the DNA, it is a __ mutation

what is repair mech. & type of mut. introduced if has donor template?

no donor template → use NHEJ, which introduces indels WITHIN the 4th codon

• is a frameshift mutation shown in the pic

donor template → use HDR, which has no mutations b/c is a homologous chromosome

---

where the gRNA sequence includes the 20 nucleotides upstream from PAM, not including PAM, but including the 3 nts b/w Cas9 cleave site & PAM

remember that translation start site is w/ ATG on the target DNA, while TSS (transcriptional start site) is at the promoter

(to introduce precise change to the genome via CRISPR-Cas9 & HDR)

give answer & explain the thought process

(~eh) when looking at the starting DNA in black, how would you do HDR via SDSA if using the top strand as the one that FIRST STARTS annealing to the repair template?

• which part would be the donor template vs 3’ overhang? explain the thought process

b/c:

donor template is similar to the target gene, so the donor template is complementary to the strand opposite of the target gene strand

SO

1. change the specific DNA strand to have the mutation

2. draw out the complementary sequence to that strand (b/c donor template is complementary to the non-target gene/strand & similar to target gene/strand

__

SDSA:

• Cas9 cleaves & forms DSB in DNA

• 5’→3’ exonuclease forms 3’ overhangs (only 1 is indicated in the pic)

• the indicated overhang will anneal to the homologous seq. on the repair template

• will be extended via DNA pol. in the 5’ → 3’ direction

• that newly synthesized strand will reanneal to its broken end

• (not known, but complementary stand gap is filled in via DNA pol using newly synth. strand as template)

look at bottom pic for donor template & 3’ overhang

---

(You use the lower strand because HDR copies from a complementary template; the ssDNA donor is designed to match that template strand (5′→3′), except at the target base where the mutation (G→A) is introduced so that repair installs the desired change.)

for SCD

you can use gene editing to treat sickle cell disease that is caused by mutations in the __ gene

• mutations in this gene will do what? (1)

t/f: nearly all sickle cell disease (SCD) patients have some type of variant that is treatable w/ gene editing

• state 1 very common SCD variant

____

normally, not when have SCD:

relate beta- and gamma-globin

what leads to the decrease in gamma-globin expression at birth? explain how

…

SO summary: people w/ SCD will express __ levels of beta-globin, but the expressed beta-globin is __/abnormal

you can use gene editing to treat sickle cell disease that is caused by mutations in the beta-globin gene (HBB)

• mutations in beta-globin gene will disrupt red blood cells that carry oxygen

true: nearly all sickle cell disease (SCD) patients have some type of variant that is treatable w/ gene editing

• A-to-G mutation in HBB (beta-globin gene/hemoglobin subunit beta gene)

____

normally, not when have SCD:

• gamma-globin expression decreases after birth

• beta-globin expression increases after birth

gamma-globin expression is turned off at birth by the BCL11A transcription factor b/c:

• BCL11A (TF) binds to an enhancer upstream of the gamma-globin gene & inhibits gamma-globin expression

…

SO summary: people w/ SCD will express NORMAL levels of beta-globin, but the expressed beta-globin is MUTATED/abnormal

for SCD

what is the COMMON gene editing treatment for SCD? (3 parts in one sentence)

• specifically, you use CRISPR-Cas9 to (1 ← which usually/normally does what?) located in (1)

• & within this enhancer, there is a …

why do you target the enhancer of BCL11A (gene)?

why not target the coding region of the BCL11A gene?

SCD treatment:

• substitute mutant beta-globin w/ gamma-globin by prolonging gamma-globin expression via eliminating BCL11A in blood cells using CRISPR-Cas9

  • specifically, you use CRISPR-Cas9 to mutate an enhancer located in the intron of the BCL11A gene (where the enhancer that usually drives BCL11A expression blood cells)

  • & within this enhancer, there is a binding site for a TF necessary for BCL11A expression in red blood cells

target enhancer of BCL11A b/c want to selectively turn off/eliminate BCL11A expression ONLY in blood cells

if you target the coding region of BCL11A, then it can cause intellectual disability b/c BCL11A is expressed in multiple cell types & tissues

for SCD

if a donor template was NOT used. how was mutation in BCL11A enhancer created?

• explain the steps/process

mutation in the BCL11A enhancer was created via NHEJ, so the indel mutation was random → which causes gamma-globin reactivation SO can treat SCD

• cut out the enhancer located w/in intron of the BCL11A gene (via…)

• NHEJ occurs to introduce a mutated enhancer / enhancer w/ indels

  • cause the TF binding site (aka GAT1 binding site) to break

  • SO enhancer stops working

  • SO BCL11A gene expression decreases

  • SO little to no inhibition of gamma-globin expression AKA gamma-globin reactivation, which will treat SCD

for SCD

what is the TAILORED gene editing treatment for SCD? (← incl. specific term & def.)

• why is b/c normally, SCD is commonly caused by (1)

overview:

• base editing involves what 2 types of base editors?

• base editors create (3), rarely create (3)

revert SCD by changing a single nucleotide via base editing ← combining CRISPR-Cas9 w/ nucleotide deaminases

• b/c normally, SCD is commonly caused by missense mutation in the beta-globin gene (from wt allele w/o SCD → mutant allele ass. w/ SCD)

overview:

• base editing involves what adenosine base editors & cytosine base editors

• base editors create:

  • single nucleotide mutations

  • mutations of coding DNA (silent, missense, nonsense)

  • mutations of non-coding DNA (promoters, enhancers, mRNA splice sites, etc.)

• base editors rarely create:

  • deletions (in general / small or large)

  • insertions (in general / small or large)

  • frameshift mutations

for SCD treatment via base editing (which is a TAILORED gene editing treatment for SCD)

purpose of adenosine base editor

steps of adenosine base editors (ABEs) (← 5)

_

why does adenine base editor (ABE) use Cas9 w/ nickase activity?

adenosine deaminase creates __ mutations

_

draw out adenosine base editing w/ A-to-G mutations

---

note: inosine does NOT pair w/ __, only pairs with (3)

targeted mutagenesis to change A into a nucleotide that does not cause SCD/disease

• adenosine deaminase will attach to Cas9 nickase

• adenosine deaminase use Cas9 nickase activity to deaminase A → I (inosine)

• Cas9 nickase cleaves ONE strand of DNA / the unedited strand

• T → C via DNA replication or BER (base excision repair), where BER uses the intact edited strand

  • (DNA repl. is cell divides, DNA pol pairs C to the I)

  • (BER is DNA mismatch repaire that adds C opposite to I)

• convert I → G during DNA replication

_

adenine base editor (ABE) use Cas9 w/ nickase activity b/c it forces the cell to use I (inosine) as template

adenosine deaminase creates A-to-G mutations

---

note: inosine (I) does NOT pair w/ guanosine, only pairs with A, C, T

…

bottom pic shows adenosine base editing w/ A-to-G mutations

(top pic is what the cell would normally do)

for SCD treatment via base editing (which is a TAILORED gene editing treatment for SCD)

def. BER

• what strand is used as template for repair

the Cas9 nickase directs the BER pathway to use the __ strand for repair, to __ the likelihood of incorporating the mutation (which neutralizes the disease allele from: mutant allele ass. w/ SCD → __ allele that __ cause SCD aka diff. from the wildtype allele)

overall, adenine base pairing __ the symptoms of SCD in a mouse model & in a patient

_

t/f: any adenosines in the gRNA could be mutated by the adenosine base editor b/c multiple codons have an A

BER — repairs changes to DNA that involve 1 or few nucleotides that don’t cause major change to DNA

• use intact edited strand as template for repair

  • (for changing I/T → I/C, where “intact” is same I & “edited” is A → I)

the Cas9 nickase directs the BER pathway to use the intact edited strand for repair, to increase the likelihood of incorporating the mutation (which neutralizes the disease allele from: mutant allele ass. w/ SCD → mutant allele that doesn’t cause SCD aka diff. from the wildtype allele)

overall, adenine base pairing reverts/reverses the symptoms of SCD in a mouse model & in a patient

__

true

from L to R is:

missense, silent, silent, missense

for SCD treatment via base editing (which is a TAILORED gene editing treatment for SCD)

for cytokine base editing, it causes __-to-__ mutations that are highly ass. with cancers

• 2 ways to reverse this mutation

^ draw what this looks like

causes C-to-T mutations, highly ass. w/ cancers

• reverse C-to-T mutations by changing:

  • T → C

  • changing A → G, then T → C

relate #’ ends of coding strand gene, #’ ends of mRNA, N or C-terminal or protein

5’ end of coding strand = 5’ end of mRNA = N-terminal of protein

• (think N-terminal has positive amine groups that DNA moves away from b/c of “-” backbone & b/c DNA pol moves from 5’→3’, then N-terminal is at 5’ end)

3’ end of coding strand = 3’ end of mRNA = C-terminal of protein

for FXS

what are the 2 CRISPR-Cas9 approaches to reverse silencing of the FMR1 gene in FXS?

__

for FXS:

• the gene that produces FMR1 is located where? it appears “fragile” b/c …

• normal alleles for FMRI has about __ repeats of the CGG sequence in a 5’ UTR near the TSS, while FXS has __ repeats

CGG repeat expansion __ gene expression, so (what happens to the protein?)

__

in premutation state of FXS, FMR1 mRNA levels __, but protein levels __

in general:

• mRNA levels increase due to __ transcription of the FMR1 gene

• if protein levels decrease, it is b/c there is __ translation of the mRNA

• use Cas9 to cleave near CGG repeats & do DNA repair to reduce the # of repeats

OR

• use an enzyme-dead Cas9 to recruit a demethylase (Tet1)

__

for FXS:

• the gene that produces FMR1 is located during in the “fragile” region of the X-chromosome; appear “fragile” b/c decreased chromatin compaction

• normal alleles for FMRI has about 54 repeats of the CGG sequence in a 5’ UTR near the TSS, while FXS has >200 repeats

CGG repeat expansion SILENCE gene expression, so FMRP/FMR1 protein is not produced

__

in premutation state of FXS, FMR1 mRNA levels increase, but protein levels decrease

in general:

• mRNA levels increase due to enhanced/increased transcription of the FMR1 gene

• if protein levels decrease, it is b/c there is stalled translation of the mRNA

for the CRISPR-Cas9 approach to reverse silencing of FMR1 gene in FXS: w/ living Cas9

what is the method involving living Cas9?

(reminder that the CGG repeats are located in the 5’ UTR of the mRNA)

which DNA repair pathway will likely be targeted? why?

• furthermore, HDR is only active in dividing cells BUT (is or is not?) active all the time in dividing cells; while NHEJ occurs in dividing & non-dividing cells, meaning that it is the most __ repair mechanism for repairing DSBs

^^^ knowing all this, state the very general steps of this CRISPR-Cas9 approach to reverse FMR1 silencing in FXS (4)

reducing the number of CGG repeats by doing Cas9 cleavage near the repeats & then DNA repair

use NHEJ

• you don’t need precise deletions or changes to reduce the # of CGGs b/c the CGGs are located in the non-protein-coding region of the gene, SO can use imprecise repair method

• furthermore, HDR is only active in dividing cells BUT IS NOT active all the time in dividing cells; while NHEJ occurs in dividing & non-dividing cells, meaning that it is the most ACTIVE repair mechanism for repairing DSBs

  • i.e. HDR is not active in post-mitotic cells, like in neurons

^^^ knowing all this, the general steps are;

• identify PAM site

• find gRNA target site (AKA design/form gRNA)

• find Cas9 cleavage site → form DSB

• then, do NHEJ repair

for the CRISPR-Cas9 approach to reverse silencing of FMR1 gene in FXS: w/ living Cas9

DNA is very/abnormally HIGHLY methylated in __ __ cells from __ patients, while DNA has low methylation in __/__ patients

these __ __ cells are treated w/ CRISPR-Cas9 to target the CGG repeats, to get:

• (1 ← 1) cells that show NO change in DNA methylation patterns

• (1 ← 2) cells show DECREASE in DNA methylation compared to FXS NPC cells, where DNA methylation is reduced to NORMAL WILDTYPE levels in these cells

__

answer 2Qs in the pic, explain

conclusion (1)

DNA is very/abnormally HIGHLY methylated in neural precursor cells (NPCs) from FXS patients, while DNA has low methylation in normal/wildtype patients

these neural precursor cells (aka both wildtype & FXS NPCs) are treated w/ CRISPR-Cas9 to target the CGG repeats, to get:

• edited wildtype cells (E1) cells that show NO change in DNA methylation patterns

• edited Fragile X cells (E1, E3) cells show DECREASE in DNA methylation compared to FXS NPC cells, where DNA methylation is reduced to NORMAL WILDTYPE levels in these cells

(^ WT NPC, WT NPC E1 vs. FXS NPC, FXS NPC E1, FXS NPC E3)

__

(top pic) no FMR1 protein is expressed in Fragile X cells

• b/c DNA methylation typically reduces gene expression, and FXS NPCs have a lot of highly methylated DNA / DNA with abnormally high amount of methylated NPCs in FXS patients

(bottom pic) there is now FMR1 protein being expressed in the Fragile X cells that were edited by CRISPR-Cas9

• b/c CRISPR-Cas9 editing of FXS NPCs to get FXS NPC E1/3 will decrease DNA methylation back to normal wildtype levels

conclusion:

• CRISPR-Cas9 can be used to edit FXS NCPs to restore FMR1 protein (restore FMR1 gene expression)

for the CRISPR-Cas9 approach to reverse silencing of FMR1 gene in FXS: w/ enzyme-dead Cas9

what is the method involving enzyme-dead Cas9?

state the steps of this CRISPR-Cas9 approach to reverse FMR1 silencing in FXS (1 step w/ 2 results)

_

answer the 2Qs in pic (don’t have to explain/self-explanatory)

use enzyme-dead Cas9 to recruit regulators for transcription and chromatin, specifically cytosine demethylase called Tet1, to reverse silencing of the FMR1 gene

• (where there can be many diff. enzymes recruited b/c many functions of transcription activator/repressor, DNA demethylase, histone acetyltransferase, methyl transferase)

steps:

• the enzyme-dead Cas9 recruits Tet1 to the FMR1 gene, where:

  • dCas9-Tet1 / dC-T will increase FMR1 gene expression

  • as a result, FMR1 protein levels increase in cells treated w/ dCas9-Tet1

__

(top pic) dCas9-Tet1 / dC-T recruited to the FMR1 gene will increase FMR1 gene expression

(bottom pic) FMR1 protein levels increase in cells treated w/ dCas9-Tet1

• whereas, FX52_mock where there’s no dCas9 or Tet1 (is just the control) shows little to no FMR1 protein expression b/c of abnormally high DNA methylation at FXS NPCs that reduce FMR1 gene expression and resulting FMR1 protein expression

CRISPR-based treatments are already showing promise in treating patients with sickle-cell disease.

SO why are CRISPR-based treatments not being used to treat patients with neurological disease at this time? (2 similar reasons)

• for 1 of the reasons, explain why it is hard (aka the process)

  • t/f: you can’t just inject Cas9 and gRNA into somatic cells & expect it to cause precise mutations in specific target genes (i.e. mutated enhancers in BCL11A gene in blood cells)

b/c unlike blood cells, you can’t isolate neurons from patients, edit the neurons’ genomes, and re-inject these edited genomes into the patient

these neurological diseases (i.e. FXS) are affecting neurons, which are mostly made in utero, SO would have to treat patients in utero which is unethical

&

b/c it's hard to deliver CRISPR-Cas9 gene editing parts like Cas9 and gRNA into cells

^ CRISPR-Cas9 targets somatic cells by:

• isolating somatic adult stem cells from the patient & injecting these cells w/ CRISPR reagents

• identify cells that have the intended/wanted gene edits & inject those specific gene-edited stem cells back into the patient

^ true: you can’t just inject Cas9 and gRNA into somatic cells & expect it to cause precise mutations in specific target genes (i.e. mutated enhancers in BCL11A gene in blood cells)

• (there are many biological, chemical, and physical methods to introduce these CRISPR-Cas9 gene editing components into somatic cells)