GBT 331 – PRINCIPLES OF BIOTECHNOLOGY I
2022/2023 SESSION
INTRODUCTION TO NEW TECHNOLOGIES IN BIOTECHNOLOGY
A) RNA Interference (RNAi)
RNAi mediated silencing is a coordinated series of sub-cellular events that ultimately lead to post-
transcriptional termination of gene expression. It is a mechanism in which RNAs are specifically
degraded if they are similar in sequence to a transgene, especially if it is designed to produce
double stranded (ds)RNA. The RNA silencing phenomenon has been termed co-suppression or
post-transcriptional gene silencing (predominantly used to describe plant and plant virus-related
silencing events), quelling (in fungi) and RNA interference (in vertebrates and invertebrates). The
mechanisms that underlie these phenomena are based on broadly similar principles, and that
comparable signaling molecules are involved.
The most important feature of RNA silencing is the cleavage of the dsRNA into “short dsRNA
fragments known as short interfering RNAs (siRNA)” of 21-25bp in length, by a ds-specific
ribonuclease termed “Dicer’ which occurs in plants as a family of 4 enzymes with different
functions. The strand of siRNA complementary to the target RNA becomes incorporated into the
RNA-induced silencing complex (RISC) leading to mRNA degradation and gene silencing. Since
dsRNA is a potent activator, any RNA virus which replicates via dsRNA intermediate will induce
PTGS in plants. Therefore, RNAi can be used as a technique that takes advantage of an endogenous
biochemical pathway, directing it to the destruction of specific target transcripts (gene silencing).
In addition, RNAi can be used to produce transgenic animals (known as knockdown animals),
resembling the null phenotype (- / -). Additionally, since RNAi may be tamed to promote silencing
efficiencies ranging from 0.1% to 100%, animals with partial phenotypes, known as hypomorfic,
may be generated. These hypomorfic individuals are very informative since they reveal
intermediary and unprecedented phenotypes and because the null genetic construction may happen
to be unviable.
Currently, most studies use RNAi as a tool for reverse genetics (identification of gene function),
but the applications are numerous: i) disease control (viruses; bacterial diseases; parasites; genetic;
tumors), ii) production of animals of commercial interest and iii) production of animal models for
,research use. Other possible future applications include: control of drug consumption, pain relief,
modulation of sleep, among many others.
Mechanism of RNAi
, B) GENOME EDITING
Genome editing (also called gene editing) is a group of technologies that give scientists the ability
to make tiny controlled permanent and heritable changes at specific sites in the genome of an
organism, mediated by the cell’s own DNA-repair machinery, and lacking in any foreign DNA.
These technologies allow genetic material to be added, removed, or altered at particular locations
in the genome. Several approaches to genome editing have been developed including
Meganucleases, Zinc Finger Nuclease and TALEN. A recent one is known as CRISPR-Cas9,
which is short for clustered regularly interspaced short palindromic repeats and CRISPR-
associated protein 9. The CRISPR-Cas9 system has generated a lot of excitement in the scientific
community because it is faster, cheaper, more accurate, and more efficient than other existing
genome editing methods.
Meganucleases
Meganucleases are endodeoxynucleases characterized by a large recognition site, DNA sequence
of 12 to 40 base pairs. This site generally occurs only once in any given genome. For example, the
18-base pair sequence recognize by the I-Scel meganuclease. Meganucleases are considered to be
the most specific naturally occurring restriction enzymes. Number of naturally occurring
meganucleases is limited and not sufficient to cover all potentially interesting loci. Construction
of sequence specific enzymes is costly and time consuming.
Zinc Finger Nucleases (ZFN)
Zinc finger motifs occur in several transcription factors. C-terminal part of each finger is
responsible for the recognition of the DNA sequence. The recognized sequences are short, made
up of around 3 base pairs, so a protein could be engineered for binding a specific DNA sequence
in the genome of around 20 base pairs by combining 6 to 8 zinc fingers for a recognition site.
These synthetic proteins could be used in editing of a specific gene by fusing it with the catalytic
domain of the Folk endonuclease. The ZFN-based technology is complex and expensive.
TALEN system developed TALEs produced by the phytopathogenic bacteria Xanthomonas genus.
TALE belong to a DNA binding protein family and can be used to activate their target genes. DNA
binding domain of the TALE proteins consist of tandem repeats of 34 amino acid residues
monomers, each of them binds one nucleotide in the target nucleotide. The final constructed
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