CRISPR Flashcards
How is genetic engineering different from genome editing?
Genome editing targets the insertions into site specific locations, unlike genetic engineering that inserts genetic material randomly into the host genome
Give three examples and three applications of random integration
Examples: Transgenesis, transposon mutagenesis, (retro)viral mutagenesis. Applications: 1) Functional studies of overexpressed genes 2) Integration of exogenous genes 3) Functional genetic screens
Give three examples of genome editing and applications
Examples: 1) Knockout alleles 2)Conditional alleles 3)CRISPR genome editing, Applications: 1) Functional studies of genes 2) Tagging of genes to study localization or expression patterns in vivo 3) Functional genetic screens
What is a pseudopregnant mouse?
I female bred with an infertile mouse that behaves hormonally pregnant allowing its use as a recipient for embryos
What are meganucleases?
Microbial endonucleases with very long (more than 14 bp) recognition sequences. Unlike other nucleases, such as restriction enzymes, meganucleases are highly specific and often recognize unique sequences that are not found elsewhere in the genome.
What can meganucleases generate?
Fusion proteins
What is the problem with meganucleases?
It is very specific, but it is unlikely that any gene of interest contains that recognition sequence
what are zink-finger nucleases?
Artificial nucleases comprising of engineered zink-finger domains and engineered catalytic subunit of Fokl endonuclease
What are zink finger domains?
they are derived from transcription factors and each domain recognizes about 3 bp
What is Fokl?
An endocnuclease with separate DNA recognition and cleavage domains, Fokl cleavage domains activate upon dimerization (with another Fokl enzyme)
What is the problem with the zink fingers?
A nuclease has to be created for each application
What are TALENs?
Transcription activator-like effector nucleases
What are transcription activator-like receptors?
Proteins secreted by a plant pathogenic bacteria
What do TALEs do?
Bind promotor sequence in host cells to activate genes that aid infection
What are the repeat domains of TALEs?
One domain contains 33-35 amino acids repeat motifs with variable aa at position 12 and 13 and binds to DNA
What is RVD?
Repeat variable disresidue
How specific are RVDs?
For single nucleotides
Why are TALEs so good in genome editing?
TALE domains can be engineered to specifically target and bind to any desired DNA sequence
What is TALEN?
Fusion proteins containing a series of repeat motifs and Fokl catalytic subunit
What is the downside of TALEN?
A new nuclease has to be created for each application
What are the DNA repair pathways that genome editing techniques rely on?
Non-homologous end-joining and homologous recombination
When does non-homologous end-joining take place and how is the error rate?
Mainly in G1. error prone
When does homologous recombination take place and how is the error rate?
in G2, precise and error free
When can cells be tricked into HR?
When providing an exogenous DNA template
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
What do Cas genes encode for?
Helicases and nucleases
What are two important CRISPR systems and their characteristics?
Type 2-single protein effector, Cas9 tracrRNA; Type 5-single protein effector; Cpf1-single RNA guided
How many classes of CRISPR?
2
What class are type 2 and 5?
2
What is guideRNA?
A fusion of crRNA and tracr RNA
What is the function of tracrRNA?
To doc into the CAS9 protein
What is Cas9?
An effector nuclease
What is PAM? And some characteristics
Protospacer adjacent motif, only present in targeted DNA and CAs 9 cuts 3 bases upstream from PAM
What is a CRISPR/Cas system?
A microbial adaptive immune system
What is the difference in genome editing?
tracrRNA and and crRNA can be fused into a single guide (sg) RNA
What are the key findings of the first reports of clustered repeats?
Clustered repeats were described in halophilic archaea, the RNA transcripts of these sequences were detected and speculations about involvement in replicon partitioning
When were the first CRISPR loci discovered?
1987
When was the function of programmable CAs 9 demonstrated in vitro?
In 2012
What was published in 2013?
The first genome editing in mammalian cells
When was the nobel prize for CRISPR awarded?
2020
What is forward genetics?
Unbiased approach to identify genes involved in a known process
What is the process when you look at a gene and then try to identify what it does?
Reverse genetics
What are the wo nuclease domains of Cas9?
RuvC and HNH
What nuclease domain cleaves the target DNA strand which is complementary to the crRNA spacer?
HNH
What does RuvC do?
Cleaves the non-complementary non-target DNA strand that contains the PAM site
What are the potential problems of Cas9?
Mismatches (more than 11 nt) upstream of the PAM can be tolerated
* Cas9 can tolerate up to 3 mismatches in the target sequence
3 to-do’s to prevent off-targets:
- Design guides with low off-target scores: few near identical sequences
elsewhere in genome - Design multiple guides (preferably targeting different exons)
- Make use of Cas9 nickase (nCas9) or high fidelity variants of Cas
What is CRISPRi?
Targeting of dCas9 fused to
transcriptional repressors (e.g. KRAB
domain) to promotor of gene(s) of
interest
What is CRISPRa?
: Targeting of dCas9 fused to
transcriptional activators (e.g. VP64) to
promotor of gene(s) of interes
What is Cas9 effector fusions?
Cas9 effector fusions refer to the engineering of the Cas9 enzyme by fusing it with additional protein domains or functional units, which can modify or enhance its activity. The effector domains can be added to Cas9 either by fusing them directly to the Cas9 protein or by linking them to Cas9 through a flexible linker.
Examples of fusion of epigenetic modifiers to dCas9:
Histone acetyltransferases, DNA demethylases, DNA methyltransferases
What do the base editors do? What do they edit and what is the effector fuser?
They edit cytosine, the fuser is cytidine deaminase that converts C into U
What can happen with G-U?
Transform to A-T by replication
What modifications are long-term and can be inherited by daughter cells?
Epigenetic modifiers
What does an adenine base editor do?
dCas0 or Cas 9 fuses to adenosine deaminase converting A to I, T-I can be converted to C-G
Explain prime editing
Cas9 nickase fuses to a reverse transcriptase; peg RNA (prime editing guide) contains a spacer sequence for targeting a primer binding site that hybridizes with the 3’ prime end of the nicked strand and an RT template containing edits
What is the advantage of prime editing?
It does not induce a DSB and the nick can be resolved by a 5’ flap cleavage results in less aerror
Which of the following is a CRISPR/Cas base editor?
A. Fusion of Cas9 with a reverse transcriptase
B. Fusion of dCas9 or nCas9 with an adenosine or cytosine deaminase
C. Fusion of Cas9 with an adenosine or cytosine deaminase
D. Fusion of dCas9 or nCas9 with a methyltransferase
B
