Winge Flashcards
Inactivation of genes has been an important tool to study the functions of
genes in many model organisms. What is the advantage of the CRISPR/Cas9
technology compared to the previous knockout methods?
The advantage of the CRISPR/Cas9 technology is that it is more precise and quicker than previous technology. The previously used zinc fingers and TAL effectors were fused to the endonucleolytic DNA cleaving domain of the Fok1 endonuclease, making ZNFs or TALENs. Since the Fok1 acts as a dimer, a pair must be designed for this to work. CRISPR/Cas9 uses a single-guide RNA which directs the Cas9 to the right site. The speed, simplicity, low cost, plus the high activity and specificity of Cas9 is why it is better.
2. Bacteria have CRISPR/Cas systems that often express a trans activating CRISPR RNA (trcrRNA), what is the function of the trcrRNA?
In the natural bacterial systems, the function of the tracrRNA is multifold. It is expressed along with pre-crRNA, and along with RNase is part of the processing of the pre-crRNA into crRNA. Then, as the 5’ end of the tracrRNA and the 3’ end of the crRNA, and together they act as a complex with Cas9 to guide the enzyme to the right site and promote cleavage.
- The Cas9 protein contain two nuclease domains, the RuvC and HNH
domains. If the catalytic sites in RuvC and HNH are inactivated the protein
cannot produce double stranded DNA breaks and is called a dead-Cas9
(dCas9). Can the dCas9 still be of any use? Describe two examples of how it
can be used.
Dead Cas9 can still be used. As the Cas protien is not part of the guide or binding, its “death” does not inhibit the binding. dCas9 bound to an mRNA will inhibit the translation of this mRNA, thus enablig studying what happens if that gene is not expressed in a cell.
Recently, a different approach, termed base editing, has been developed to introduce specific nucleotide changes to the genome. By fusing a cytidine or adenosine deaminase enzyme to a catalytically impaired Cas9 [dead Cas9 (dCas9)], targeted conversion of a C:G to T:A or A:T to G:C base pair can be facilitated. However, this can act on any C or A within 5 nts, and is not as specific and is currently a limitation for how much it can be used.
- Bacteria uses CRISPR/Cas systems to protect themselves from invading
bacteriophages. This has led to an arms race where the bacteriophages try to
evade the “CRISPR defence”. In this process some bacteriophages express
anti-CRISPR proteins that inhibits Cas9. Can anti-CRISPR proteins have any
useful applications in biotechnology?
Anti-CRISPR molecules can be used as an alternative to antibiotics, with phage therapy instead. The phage only infects the desired bacteria causing the infection, thus avoiding disturbing the microbiome in the gut (and C. difficile takeover). Another use is to reduce off-target effects, which are more common if there is too much CRISPR/Cas in ratio to the amount of binding sites.
- How the CRISPR/Cas system are delivered to eukaryotic cells may vary from
organism to organism. What are the challenges related to this delivery
process? How do we produce or engineer cells that contain Cas proteins and
guide RNAs?
Delivery methods for CRISPR-systems can be done using viruses or plasmids. Plasmids are common in prokaryote lines, but can induce an immune response in eukaryotes, in addition to the delivery efficiency generally being low. Viral vectors can be used to transfer the plasmid directly into the nucleus of the eukaryotic cell, where it is safer from the immune system. However, this method does not allow unlimited size of the fragments. One potential solution is to inject the gene in more than one plasmid, and have them join to the full gene inside the nucleus.
Cells that contain Cas proteins and guide-RNAs can be made by inserting a plasmid with a sg-RNA ans the correct Cas protein (if it is not already native to the cell). It is important that the vector is expressible in the cell, including the right promoter, origin of replication, termination, etc.
