CRISPR/Cas in eukaryotic cells and organisms Flashcards
Genome editing (in general)
The ability to make precise changes to a specific locus in the genome
Zinc finger nucleases (ZFNs)
Fusion proteins comprised of DNA-binding C2H2 zinc fingers fused to the Fok1 nuclease (DNA cleaving domain).
Rely on protein-DNA interactions for DNA targeting.
Labor intensive
Transcriptional activator-like effector
nucleases (TALENs)
Chimeric proteins comprised of a
“custom made” DNA-binding domain
(33-35 a.a. repeats) fused to the Fok1 nuclease domain.
Rely on protein-DNA interactions for DNA targeting.
Labor intensive
Construction of CRISPR
Repeated unit of 21-40bp
Spacers of 20-58bp
Homologous to sequences of bacteriophages, prophages and plasmids.
Components needed for genome editing by CRISPR/Cas9
Cas9: CRISPR-associated protein 9 nuclease (Streptococcus pyogenes)
(single) Guide RNA (crRNA + tracrRNA ~100 nt), which include the DNA target sequence at 5’-end.
crRNA = CRISPR RNAs tracrRNA = trans-activating crRNA
Single guide RNA (sgRNA) can be constructed by fusing a crRNA containing the targeting guide sequence to a tracrRNA that facilitates DNA cleavage by Cas9 nuclease.
DNA cleavage and rejoining by CRISPR/Cas9 system
PI domain of Cas9 recognizes the NGG PAM-site adjacent to the target site.
Produces double stranded DNA breaks, which the cell try to repair.
Without a supplied repair template the cell uses the error prone Non Homologous EndJoining pathway (NHEJ).
If template: homologous recombination => more precise.
Design of sgRNA complementary to the target site
Select the target region or gene which you want to edit (knock-out).
A software program finds PAM sites (ex. NGG) of both DNA strands selected.
The program evaluate the target sites. • Similarities to other genomic regions • GC content • Secondary structures • Avoid poly-T regions if RNA polIII is used to generate the sgRNA.
You receive a list of potential good target sites.
- Need to check for SNPs and indels (mutations) in you target sequence.
Order oligonucleotides for target site (used to produce adapters)
Homologous recombination (HR) vs Non-homologous end joining (NHEJ)
Off-target effects with CRPSPR-Cas9
Unintended point mutations, deletions, insertions inversions, and translocations.
Reason: cut at unintentional sites.
Enhancing specificity using nickases.
Instead of producing ds breaks, the nickases targets only one DNA strand.
SS DNA breaks are normally repaired without problems by the DNA repair apparatus.
If the two target sites are close to each other (20-50 bp), it is
possible to induce NHEJ and produce indels at a specific loci without off-target effects.
Cas9 domains
Cas9 with both domains (RuvC and
HNH) intact will make DNA double
strand breaks.
Cas9 nickase with mutation in RuvC: Cleaves only the DNA strand that is complementary to gRNA.
Cas9 nickase with mutation in HNH: Cleaves only the DNA strand that does not interact with gRNA.
Inactive (dead) dCas9, with mutations in both nuclease domains.
Can be converted into a general DNA-binding domain => enables tagging with other proteins.
Prime edit (PE) by pegRNA
pegRNA : prime editing guide RNA
The nickase (Cas9) makes a cut in the DNA (one strand). A part of the pegRNA is complementary to DNA flanking the nick site. The reverse transcriptase transcribes this into DNA, which is used as a repair template and through homologous recombination repair the DNA break.
Efficiency of PE have been up to 30%
Photoactivatable CRISPR/Cas9 for optogenetic genome editing
The phage-derived anti-CRISPR (Acr) protein inhibit type II CRISPR systems.
The LOV2 domain is blue-light-induced (changes conformation in blue light).
The LOV2 domain is fused to Acr and expressed together with Cas9.
The N and C termini of LOV2 are in close proximity in the dark, keeping Acr in a native (active) conformation.
Photoexcitation with blue light results in unfolding of the LOV2 terminal helices and induce a conformational change in Acr
resulting in its inactivation, no Cas9 binding.
=> Cas9 is activated.
Gene drive (mutagenic chain reaction)
A method to increase editing of both gene alleles and convert them to the same type.
Process that promotes or favors the biased inheritance of certain genes from generation to generation.
