Lecture 9 - Genetic Medicine Flashcards
can be used to remove, change, and add DNA after cutting it
CRISPR/Cas9
early models of genome editing performed the same functions as current ones, but the largest difference is:
the newer ones (like CRISPR-Cas9) are easier to program and more efficient
when was the CRISPR sequence discovered and how?
1987, discovered that the bacteriophage genome had strage repeat sequences but at the time their function was unknown
in 2009, it was found that the repeat CRISPR sequences function in:
protecting against viruses
in 2012, it was found that Cas9:
works alongside CRISPR to target and cut specific DNA systems
what does CRISPR-Cas9 stand for?
- CRISPR = clustered regularly interspaced short palindromic repeats
- Cas9 = CRISPR associated protein 9
CRISPR-Cas9 is part of the prokaryotic immune system that:
defends against viruses
what is the purpose of guide RNA?
binds to virus sequences
how does the guide RNA/Cas9 complex work?
binds to invading DNA and cuts it up
Cas9 is recruited to the DNA target site by the duplex:
tracrRNA:crRNA (guide RNA)
binds the complementary DNA upstream of the PAM sequence
crRNA
what is the PAM sequence?
protospacer adjacent motif
what are the three key elements to the CRISPR-Cas9 system?
1) the Cas9 DNA endonuclease
2) crRNA
3) a trans-activating crRNA (tracrRNA)
contains a 20bp sequence complementary to the target
crRNA
acts as a bridge between the crRNA and the Cas9 system
trans-activating crRNA (tracrRNA)
DNA endonuclease is targeted to a DNA sequence via a ______, upstream of the ______, resulting in a ______ upstream of the NGG
a single guide RNA (sgRNA) sequence, protospacer-associated motif (PAM), bp double-strand break (DSB)
using a CRISPR-Cas9 system, the resulting double-strand breaks are sealed back together by either ____ or ____
non-homologous end joining (NHEJ), homology-directed repair (HDR)
what is a challenge to using CRISPR-Cas9?
off-target DNA cleavage
the 3’ end of the DNA target sequence must have a:
proto-spacer adjacent motif (PAM) sequence (5’-NGG-3’)
where does the Cas9 nuclease cleave the DNA relative to the PAM sequence?
approximately three bases upsteam of the PAM
the PAM sequence itself is absolutely required for cleavage, but is NOT part of:
the sgRNA sequence
how can Cas9 be delivered?
as a DNA or mRNA molecule encoding for the Cas9 gene, or it may be delivered as a functional ribonucleoprotein (RNP)
what is the challenge with delivering Cas9?
delivering the cargo across the cell membrane
what are the three elements required for base editing?
1) a Cas nickase or Cas fused to a deaminase that makes the edit
2) a gRNA targeting Cas to a specific locus
3) a target base for editing within the the editing window specified by the Cas protein
what does base editing do?
changes a single base pair in the DNA sequence
uses an engineered reverse transcriptase fused to Cas nickase and a prime-editing guide RNA to change a base pair in the DNA sequence
prime-editors
catalytically inactive, or “dead”, Cas9 (dCas9), in which point mutations evade DNA cleavage by the HNH or RuvC domains, can be repurposed as a _____ or _____
transcription activator (CRISPRa), transcription repressor (CRISPRi)
what does CRISPRa do?
a transcription activator which increases gene expression
what does CRISPRi do?
a transcription repressor which knocks down gene expression
the dCas9-p300 system is used for:
epigenetic activation
dCas9 fused to the core catalytic domain of p300 can:
acetylate target sites in the genome
in the dCas9-p300 system, acetylation of the target gene synergizes with the action of:
transcription factors and RNA polymerase II, resulting in transcriptional upregulation
CRISPR is capable of making highly specific, permanent _______ that are more likely to reduce _______
genetic modifications, target gene function
CRISPR has already been used extensively to screen for novel genes that regulate known phenotypes, including:
resistance to chemotherapy drugs, resistance to toxins, cell viability, and tumor metastasis
go review slide 268
you got this babe
the single cut CRISPR strategy restores the ______ protein
dystrophin
diseases of the eye and ear are being treated by CRISPR-Cas9 ____ in clinical trials
in vivo (directly in the living eye/ear)
type of CRISPR-Cas9 therapy which is useful for blood diseases like sickle cell anemia, hemophilia, and CAR T cancer
ex vivo therapy
what are somatic cells?
any cell in a living organism other than a reproductive cell
what are germline cells?
cells segregated and involved in the reproductive process that are passed onto the progeny
what are some ethical and legal concerns when it comes to using CRISPR-Cas9?
- safety: if gene editing specificity is imperfect, should we still use it?
- should human gene editing be allowed?
- should parents be allowed to edit the genomes of their unborn children
