CRISPR Flashcards
1
Q
What is CRISPR
A
- clustered regularly spaced palindromic repeated
- CAS = CRISPR associated proteins
- CRISPR and CAS found in 50% of bacterial species and 90% of archea
- first discovered in 1987 but function unknown until 2005 then shown to be a defence mechanism against foreign DNA molecules
- similar to RE but more complex in function
- designed to target specific DNA molecules
- CRISPR-CAs systems have many version but CRISPR-Cas9 focused on primarily
2
Q
CRISPR-Cas9
A
- Cas genes encode CRISPR-associated proteins
- which have endonuclease activity
- CRISPPR contains unique spacer DNA from invading phases between short 23-47bp palindromic repeat DNA sequences
3
Q
Stage 1: foreign DNA acquisition (aka adaption)
A
- invading bacteriophage inject foreign DNA into cell
- foreign DNA cut into short segments
- DNA segments are added into bacterial chromosome between repeated palindromic sequences (protospacers)
4
Q
Stage 2: expression
A
- cas genes are transcribed and translated to Cas proteins
- CRISPR locus transcribed to pre-crRNA
- Pre-crRNA cleaved by CAs proteins and form hairpins and unique short segments of foreign DNA = crDNA
5
Q
Stage 3: interference
A
- rRNA and trans-activating crRNA are incorporating into Cas protein and allow targeting to DNA sequence that match the unique spacer RNA
- recognize DNA sequence that it has encountered before
- binds and uses Cas endonuclease activity to cleave invading DNA
- presence of protospacer-adjacent motif (PAM) is required in target DNA
- PAM is a short weak consensus sequence
6
Q
Doudna and Charpentier
A
-in 2012 they showed how CRISPR-Cas9 can be co-opted for genome editing and engineering
7
Q
Genome auditing with CRISPR-Cas
A
- single guide RNA (sgRNA) replaces natural crRNA and tracrRNA
- sgRNA designed to target a specific sequence in genome
- sgRNA assembles with Cas9 protein to form effector complex
- 20b 5’ portion of sgRNA base pairs to complementary target sequence in genome (8-12b seed is most important
- presence of PAM near seed sequence is essential
- effector complex must bind with PAM then Cas9 unwinds DNA nearby
- if target sequence is present, sgRNA binds with it
- Cas9 makes double stranded cut in genome
- cellular DNA repair mechanisms engaged
8
Q
Cellular DNA repair mechanisms
A
- Broken ends can be rejoined without any template
- nonhomologous end joining (NHEJ)
- frequently results in small insertion or deletions which tend to disrupt gene function
- Broken ends can be rejoined using a template
- homology directed rejoining HDR
- template could be the other chromosomal copy, or donor DNA molecules provided by researcher
9
Q
NHEJ
A
- nonhomologous end joining
- most common type of repair to double strand breaks
- double strand breaks lead to problems with replication, inversions, deletions, duplications, translocations
- no template used
- nucleotides may be randomly inserted or deleted as the cleaved ends of the chromosome are rejoined
- often results in INDELS
- resulting frameshift leads to non-functional alleles —> gene silencing —> knockout
10
Q
Homology directed repair
A
- HDR
- uses same repair enzymes as in crossing over or recombination
- can use homologous chromosome as template
- in CRISPR experiments, can inject donor DNA at same time as Cas9-CRISPR to stimulate HDR
11
Q
Advantages of CRISPR-Cas9
A
- relatively cheap and easy
- targeting: can design sgRNA for any sequence
- long recognition sequences of 8-12 nucleotides in seed means more specific targeting then REs
- can use indels created by NHEJ to create gene knockout or determine gene function/phenotype
- can be introduced to intact, living cells
- can introduce Cas9 with donor DNA to stimulate HDR
12
Q
Disadvantages of CRISPR-Cas9
A
- off-target effects - cleavage sometimes no specific
- depends on cell type
- depends on normal function of repair pathways
- edited Cas9 structure for more complementary binding to DNA, but slower acting
- Germline cells have enhanced homology-directed repair (repair cleaved alleles by matching homologous chromosomes that were not cleaved
- mosaicism: not all cells edited, different genomes, so get mosaic effect
- common in multicellular embryos
- delivery of Cas9 not 100% for all cells
13
Q
Gene correction in S-phase injected human zygotes
A
- tested ability to correct a harmful mutation in pre-implantation human embryos
- injection of CRISPR-Cas9 into normal zygote S-phase caused no harm
- injection into zygotes with mutation resulted in mosaic embryos
- some cells remained untargeted mutant
- some repaired via NHEJ
- some repaired via HDR
14
Q
Gene correction in M-phase oocyte vs S-phase zygote
A
- injection of CRISPR-Cas9 along with mutation carrying sperm during M-phase in oocyte resulted in more successful production of repaired embryos
- most repaired embryos produced by HDR, and some by NHEJ
- use of guide DNA increased proportion of embryos produced by HDR from 68% to 78%
15
Q
Potential uses of CRISPR-mediated genome editing
A
- basic research (create gene knockouts)
- disrupt genes to determine unknown gene function
- editing genomes to meet human needs/desires
- donor organs from animals
- improved farm animals
- domestication of new plants for agriculture
- de-extinction of extinct species
- gene drives to eliminate insect-spread diseases
