Gene Activation, Overexpression, Inactivation, and Interference

Assigning Function by Gene Activation and Overexpression

  • Gene Inactivation by Genome Editing
    • Most efficient way to inactivate genes.
    • Utilizes genome editing with a programmable nuclease.
      • Nuclease directed to a specific site in the genome where it creates a double-stranded cut (ds-cut).
      • The cut stimulates a natural repair process called non-homologous end-joining (NHEJ) in eukaryotes, which re-joins the DNA strands.
      • NHEJ is error-prone, often leaving a short insertion or deletion (indel) at the repair site.
      • If the repair occurs within a gene, the nucleotide sequence change will inactivate the gene, resulting in error-prone gene editing.
  • CRISPR-Cas9 System
    • Most prominent genome editing system.
    • Uses Cas9 endonuclease, which is a component of the prokaryotic immune system known as clustered regularly interspaced short palindromic repeats (CRISPR).
    • The endonuclease is directed to the target site by a 20-nucleotide guide RNA (gRNA) sequence.
    • The RNA-binding site must be immediately upstream of a protospacer adjacent motif (PAM).
  • CRISPR-Cas9 Experimental Requirements
    • Requires a cloning vector to introduce the Cas9 gene and the DNA sequence for the gRNA sequence into the cells of the target genome.
    • To ensure an active endonuclease in the eukaryotic host:
      • Use an artificial gene based on the sequence of the naturally occurring bacterial Cas9 gene but utilize preferred codons for the eukaryotic host.
      • Include sequences specifying nuclear localization signals.
    • It is also possible to edit a genome by introducing a ribonucleoprotein complex comprising Cas9 and gRNA.
  • Specificity of Editing Process for Target Sequence
    • The expected frequency of a 20-nucleotide motif (length of Cas9 gRNA) in DNA sequence is once every 420=1.1×10124^{20} = 1.1 \times 10^{12} base pairs, which is approximately 350 times the length of the human genome.
    • It is unlikely that a second exact version of the target sequence occurs in the genome unless the gRNA was poorly designed and hybridizes to a repeat sequence.
    • The Cas9 system does not require complete base pairing between the gRNA and genomic DNA (gDNA).
    • Off-target editing has been detected at sites where the DNA-RNA heteroduplex had up to five mismatched positions.

Gene Inactivation by Transposon Tagging and RNA Interference

  • Transposon Tagging
    • A method used to disrupt a gene to study its function.
    • Inactivation is achieved by inserting a transposable element (TE) or transposon into the gene.
      • Most genomes contain TEs, but while many are inactive, some can still transpose to new genomic positions.
      • Normally, transposition is a relatively rare event.
      • Recombinant DNA technologies can be used to create modified transposons that can transpose in response to external stimuli.
    • Examples include the yeast transposon Ty1 and the endogenous Drosophila transposon called the P element.
  • Challenges with Transposon Tagging
    • Difficult to target individual genes because transposition is a random event, making it impossible to predict where a transposon will end up.
    • To inactivate a specific gene, a substantial number of transpositions must be induced, and all resulting organisms must be screened to find one with the correct insertion.
    • More applicable to global studies of genome function.
  • RNA Interference (RNAi)
    • A series of natural processes by which short RNA molecules influence gene expression in living cells.
    • In genomics research, it provides a means of silencing target gene expression, not by disrupting the gene itself, but by destroying its mRNA.
    • Process:
      • Short double-stranded RNA (dsRNA) molecules with sequences that match a target mRNA are introduced into the cell.
      • These dsRNAs are broken down into shorter molecules that induce degradation of the target mRNA.
        • Caenorhabditis elegans is a model organism with approximately 20,000 genes.
  • Challenges with RNAi
    • Does not always result in complete silencing of the target gene; silencing is often incomplete (referred to as "knockdown" rather than "knockout").
      • May not be possible to assess the effect of gene knockdown on the phenotype.
    • Interfering RNAs are so short that off-target effects are possible.
      • These occur when interfering RNAs bind to mRNAs other than the intended targets, leading to the silencing of more than one gene.
    • In mammals, the artificial introduction of dsRNA often results in the activation of signaling proteins called interferons.
      • Interferons stimulate an antiviral defense process displayed by both cultured cells and entire organisms.
      • The interferon response may result in phenotypic changes that mask the specific changes occurring due to target gene silencing.
      • Mouse oocytes lack an interferon response.