L13-Genome Editing Flashcards
how many base pairs are in the human genome
3 billion base pairs
what is genome editing
a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases
what does genome editing lead to
i. enabling specific targeting of sequences within the genome without impacting the rest of the genome sequence
ii. Potential to cure genetic diseases in a patient specific manner
what is CRISPR?
Clustered Regulatory Interspaced Short Palindromic Repeats
what is Cas
CRISPR associated proteins
CRISPR-Cas9 complex is part of the Adaptive immune system of prokaryotes
True
CRISPR-Cas9 complex is Found in more than 40% of bacterial genomes
true
what are the components of CRISPR-Cas9 complex
1 Cas9 - Protein component
2 crRNA – RNA component
3 tracrRNA – RNA component
What does CRISPR-Cas9 complex do
cleaves invading DNA to prevent re-infection by viruses
how does CRISPR act as an ‘immune system’ in prokaryotes against invading DNA/RNA
1- Invading DNA recognised and cut by Cas1-Cas2 protein complexes into fragments termed protospacers
2- Protospacers integrated into CRISPR locus located in the bacterial genome
3- Upon viral reinfection, transcription of the protospacers to RNA is activated which bind to Cas9
4- Cas9/RNA duplex is recruited to complementary sequence on the invading strand of DNA
5- Cas9 cuts DNA strands creating a double strand break to prevent infection
what are the components of the CRISPR locus
Transactivating RNA
Operon of cas genes encoding Cas protein components (e.g. Cas1/Cas2/Cas9)
Identical repeat array
Spacer of invading DNA
what is the guide (gRNA)
The complex formed between the Transactivating RNA (tracrRNA) and the
Protospacer/CRISPR RNA (crRNA)
what does guide RNA (gRNA) do
enables selective binding of Cas9 to invading DNA sequences
what is the function of the cas operon
encodes Cas proteins required for DNA cleavage
what do Protospacer Adjacent Motifs (PAM) do
enable Cas9-mediated DNA cleavage
what are the features of PAM
2-8 base pair sequence 3-4 base pairs downstream of the cut site
Cas9 will not cut invading DNA without a PAM site irrespective of Cas/gRNA binding
PAM sequences are not present in the CRISPR locus
Prevents bacterial CRISPR locus being targeted by Cas proteins
what RNA is bound to cas9 and what RNA is does it contain?
gRNA contains crRNA and tracrRNA
what enables site-specific cleavage thro nuclease activity
Deposition of the Cas9/gRNA complex at a desired locus of the genome
what enables specific genomic edits to be introduced
The repair of the DNA break by endogenous DNA repair pathways
where should the proto-spacer sequence (target sequence) of the gRNA be located
upstream of the PAM site
why should the gRNAs be selective to a single genome locus
to avoid off target effects
what are the mechanisms of repairing DNA
Homology-directed repair (HDR)
Non-homologous end-joining (NHEJ)
what is produced when Cas9 cleaves DNA
a double strand break
where do HDR and NHEJ function
downstream
which DNA repair mechanism enables error-prone DNA repair
NHEJ
how is DNA repair by NHEJ error prone
introduces insertions or deletions (indels) into DNA
Impacts gene function
which DNA repair mechanism enables precise DNA repair
HDR
when does precise DNA repair take place
S phase of the cell cycle
what is used in precise DNA repair
sister chromatids
what are the steps of CRISPR-mediated gene knock out via NHEJ
Target Cas9:gRNA complex to gene of interest
DSB introduced
Cell repairs the break via NHEJ
- Error prone
Indels introduced generating a frameshift
- Premature stop codons introduced
Normal gene product not expressed
what does error prone DNA repair introduce
indels
what do indels generate
a frameshift
what are the steps of CRISPR-mediated gene knock-in via HDR
DSB introduced by Cas9:gRNA complex
Introduce a template that the cell will use to repair the DSB through HDR
HDR template requires >60 bp homology arms on either side of the point of mutation/insert
PAM sites are removed from HR template to prevent re-targeting of region
Inserts of several kilobases are possible
what drives prostate cancer progression
androgen receptor signalling
what does current treatment of prostate cancer aim to
inactivate AR by blocking ligand binding
what is castrate resistant prostate cancer
an incurable form of prostate cancer
what are the 2 CRISPR based studies regarding prostate cancer
- To generate a Cas9-expressing prostate cancer cell line to knock-out AR : Knock-out strategy
- To create a modified prostate cancer cell line to study function of aberrant forms of the androgen receptor: Knock-in strategy
how is the AR gene knocked out to treat prostate cancer
a stop codon is introduced into the gene using specific Cas9 cleavage event through NHEJ error-prone DNA repair
how are androgen receptor variants produced
by alternative splicing
when are androgen receptor variants produced
when AR-targeting agents, such as enzalutamide, are present
what is the target site for enzalutamide binding
the ligand-binding domain (LBD)
what do AR-Vs lack
exons 4-8 which encodes the ligand-binding
domain (LBD)
what does Loss of the AR LBD create
constitutively active
transcription factors that are refractory to enzalutamide
what happens to the Expression of AR-Vs in advanced disease
its elevated
what results in using CRISPR knock in strategy on AR gene
create an AR-V-only expressing cell line (blocking expression of full-length AR)
what is full length AR encoded by
exons 1-8 of the AR gene
what is gRNA designed for in AR gene knock in
exon 5 (LBD)
what causes the point mutation in exon 5 of the AG gene
HDR
what does the point mutation in exon 5 of the AR gene result in
a stop codon
how does the introduced stop codon affect the AR in CRISPR knock in
stops the formation of FL-AR
what is the name of the cell line that expresses both full-length AR and AR-Vs
CWR22Rv1
what is the name of the cell line that expresses AR-Vs
CWR22Rv1-AR-EK
what protein is involved in therapeutic vulnerability in prostate cancer cells expressing AR-Vs
PARP1/2
what considerations should be in mind with regards to cell therapy
Efficacy of delivery
Regulatory guidelines
Mosaicism
Specificity - off target effects
Immunogenicity
Germline Vs Somatic
what are the methods of delivering CRISPR in the clinic
in vivo
ex vivo
how is CRISPR delivered in vivo
- Package CRISPR/Cas in a delivery vehicle
- Deliver to patient
how is CRISPR delivered ex vivo
- Remove cells from the patient / donor
- Edit genome
- Screen / expand cell populations
- Engraft cells back into patient
what is the story of the berlin patient
1995 diagnosed HIV positive, 2006 diagnosed with acute myeloid leukaemia
2007 and 2008 patient received hematopoietic stem cell transplant
Stem cell donor was homozygous CCR5Δ32
Patient no longer requires anti-viral therapy
Individuals with homozygous CCR5Δ32 have a natural resistance to HIV infection
True
what percentage of the white northern European population is homozygous CCR5Δ32
~1%
Individuals heterozygous for the CCR5Δ32 allele have a delayed disease progression
true
what percentage of the white northern European population is heterozygous for CCR5Δ32
~15%
what does CCR5Δ32 result in
32 bp deletion of the coding sequence resulting in frameshift and unstable protein
how does CRISPR editing of CCR5 confer HIV-1 resistance in vivo
Long term CCR5 disruption was observed
CCR5 disrupted HSCs were able to reconstitute a functional immune system
Viral titre reduction and increase in CD4+ T cells demonstrated HIV resistance
why cant CRISPR editing of CCR5 confer HIV-1 resistance in humans
transplanted stem cells were unable to provide HIV resistance Due to low amounts of modified cells
