Ch. 20 - Genome Defence, Genome Editing, CRISPR/Cas Flashcards
Downregulation using antisense RNA
Antisense RNA (made using the sense strand of DNA as template) is complementary to mRNA, which is sense RNA. Therefore, antisense RNA can hybridize with the mRNA, blocking translation of it. This causes downregulation of the gene in which the mRNA contains.
Antisense RNA regulation of a gene
Example: downregulation of bacterioferritin (bfr, encodes a iron-binding protein).
The bacterial chromosome contains genes for both bfr mRNA and anti-bfr mRNA. If both mRNA molecules are made, the anti-bfr mRNA pairs with the bfr mRNA and prevents it from being translated. When iron is plentiful (and the bacteria needs iron-binding proteins), the anti-bfr gene is not expressed, and only the bfr mRNA is translated to yield bacterioferritin.
RNAi, siRNA, RISC, Dicer
RNA Interference is a (eukaryotic) mechanism for gene silencing that is induced by the presence of double-stranded RNA (dsRNA). RNAi is sequence specific and involves degradation of both the dsRNA that triggered the response, and ssRNA molecules, usually mRNA, that are homologous in sequence to the triggering dsRNA.
RNAi destroys mRNA (gene silencing) that has the same sequence as dsRNA detected in the cell.
Intruding dsRNA is recognized as foreign by RDE-4 and other proteins. A ribonuclease called a Dicer cleaves the dsRNA into segments of 21-23 nucleotides with one or two base overhangs, called short interfering RNA (siRNA). This is recognized by proteins of the RNA-induced silencing complex (RISC). The RISC complex separates the two strands of the siRNA. Finally, RISC cleaves target RNA that corresponds to the siRNA.
Micro RNA (miRNA)
Small regulatory RNA molecules of eukaryotic cells from the cells/organisms own genome. miRNA are short RNA molecules that share several properties in common with siRNA, but regulates gene expression (1-200 genes) in the eukaryotic cell itself rather than acting as a defense mechanism against intruders.
miRNA is made by processing a larger precursor that folds into a stem loop. Processing occurs in two steps using the nucleases Drosha and Dicer. After binding to miRISC and strand separation, one strand of the miRNA binds to the target mRNA and prevents translation by forming a loop in the mRNA.
Dicer
Ribonuclease that cleaves dsRNA into 21-23 bp long fragments called siRNA or miRNA. Dicer has multiple domains, including a dsRNA-binding domain to hold the target mRNA, a PAZ domain that binds to the 3’-nucleotide overhang on the target, and two RNase III domains that cut 21-23 nt siRNA.
RNA-dependent RNA polymerase (RdRP)
RNA polymerase that uses RNA as a template and is involved in the amplification of the RNAi response. RNAi is extremely potent, less than 50 molecules of siRNA can silence target RNA that is present in thousands of copies per cell. This results from creation of more siRNA copies via RdRP.
In RNAi, the RISC complex cleaves the target RNA into anomalous RNA, which is used as a template by RdRP to generate more dsRNA. This is then converted into more siRNA by Dicer. This results in an amplification of the response, thus a stronger defense against “foreign” RNA.
Experimental induction of RNAi
RNAi can be used to investigate gene function in animals and plants. Small interfering RNA (siRNA) can be created artificially by looking at the sequence of a particular gene. When the dsRNA (siRNA) is injected, the gene of interest is no longer expressed, and the resulting phenotype can be assayed.
Inducing RNAi can be done in different ways. Either by injecting long dsRNA that will be cleaved into siRNAs by the Dicers, or antisense RNA that will create dsRNA with corresponding mRNA, or short dsRNAs that work as siRNA directly. Experimentally, it is more convenient to provide a DNA construct that generates dsRNA in vivo. There are three main ways to do that:
A: antisense/sense hairpin, a DNA segment with a single promoter, containing both the antisense and sense sequence, generates a dsRNA with stem and loop structure.
B: double-promoter construct, a DNA fragment flanked by to opposing promoters, going opposite ways, the two RNAs are therefore complementary and forms dsRNA.
C: two separate genes with complementary sequences, two DNA segments with one promoter each, the complementary transcripts form dsRNA.
CRISPR: Anti-viral defense in bacteria
Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR). The CRISPR system consists of a memory bank of hostile virus sequences plus a mechanism for identification and destruction of incoming virus DNA or RNA.
The CRISPR memory bank is found at the CRISPR locus, and contains repeated sequences separated by spacers. The spacers are homologous to protospacer sequences of bacteriophages and other viruses. These spacers are used to recognize foreign intruders, and are transcribed into CRISPR RNA (crRNA) when the CRISPR locus is transcribed.
Association of crRNAs and CRISPR associated (Cas) proteins promotes degradation of complementary nucleic acids. crRNA work as guides (gRNA) for the Cas protein for RNA-guided targeting of viruses.
CRISPR/Cas system
The CRISPR/Cas9 system is a prokaryotic RNA-guided defense system. There exist three types of CRISPR/Cas systems in bacteria, Type I, II and III. Type II mechanism:
When a bacteria is infected with a virus, Cas protein complexes cleaves it, yielding spacers. These spacers are incorporated into the genome at the CRISPR locus. This locus can be transcribed, yielding crRNA. A later infection by the same or similar virus/pathogen will be detected and recognized, and the crRNA will guide the Cas protein to degrade it. It acts like an adaptive immune system against foreign DNA in bacteria.
The crRNA bind to the complementary, viral DNA, and targets DNA if a protospacer adjacent motif (PAM) (e.g. NGG) is present. The Cas protein then cleaves and thereby degrades the viral DNA.
CRISPR/Cas9 system for gene editing
Single guide RNA (sgRNA) can be constructed
by fusing a crRNA containing the targeting
guide sequence to a trans-activatingcrRNA (tracrRNA) that facilitates DNA cleavage by Cas9 nuclease.
Components needed for genome editing:
Cas9: CRISPR-associated protein 9 nuclease
The Cas9 from S. pyogenes recognizes the NGG PAM-site adjacent to the target site.
Single Guide RNA (crRNA + tracrRNA ~100 nt), which include the DNA target sequence at 5’ end.
By designing the sgRNA with sequences complementary to the sequence you want to edit (target sequence), and using a PAM sequence adjacent to it, you can guide Cas proteins to cleave and edit target genes. This can be used to knock out target genes.
When the Cas9 nuclease produces double-stranded DNA breaks, the cell tries to repair it. This can be done either by Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). However, it is not known which pathway it will follow. Gene editing by CRISPR/Cas can therefore be un-specific.
Effects of CRISPR/Cas
One potential problem with the CRISPR/Cas9 technology is that the Cas9 ribonucleoprotein (Cas9 with bound sgRNA) in some instances makes cut in the DNA at unintentional sites.
This often mentioned as off-target effects.
Non Homologous End Joining (NHEJ)
Without a supplied repair template the cell uses the error prone Non Homologous End Joining pathway (NHEJ). dsDNA breaks repaired through the NHEJ pathway produce indels.
When there is a double-stranded break in the DNA, enzymes try to make compatible ends by adding or deleting nucleotides at the ends. In other words, this may lead to insertions or deletions (indels), or both, in the DNA sequence.
Homologous Recombination (HR)
HR, also called Homologous Directed Repair (HDR) occurs when there is a repair template available. This could be the sister chromatid, or in inserted template. The cut DNA is matched to a repair template. When using a sister chromatid, there may be no change. When using an inserted template, a target change can be made. Homologous recombination may induce precise genomic changes.
Zinc-finger nucleases
The DNA recognition site of zinc finger TF fused with FokI nuclease. Nucleases containing a DNA-binding domain (3 bp binding) and a DNA-cleavage domain. Cleaves un-specific. Early method for gene editing.
TALENs
Transcription Activator-Like Effector Nucleases, 33-34 aa DNA binding domain. DNA-binding domain fused with FokI nuclease. Each module recognizes a single base. Cleaves non-specific. Early method for gene editing.
