An Introduction to Genetic Modification

Playing God? – An
Introduction to Genetic
Modification
ETHAN SUNG – DOCTOR’S SOCIETY – 3/11/17
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Editing Techniques : History of CRISPR-Cas9
 First discovered by Yoshizumi Ishino, who accidentally cloned part of
CRISPR when researching the iap gene
 Found unusual repeated sequences of nucleotides interspaced throughout
DNA, the purpose was not yet known
 In 2006, scientists hypothesised that CRISPR and associated Cas genes
created bacterial immunity
 2008, discovered that regions of CRISPR corresponded to nucleotide
sequences of invaders (spacers)
 Cas9 protein observed in Streptococcus, uses CRISPR-RNA to guide to
target DNA and trans-activating RNA to cut, fused together to create a
locating/cutting tool
 2015, testing in tripronuclear zygotes showed CRISPR could cut the HBB
gene when reprogrammed, but caused mutations due to off-target cuts
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Editing Techniques :
Mechanics of CRISPR-Cas9
 Guide-RNA – composed of CRISPR RNA and trans-activated
CRISPR RNA to produce a hairpin loop active site
 PAM – small DNA sequence following the target, allowing Cas9
to bind at NGG nucleotides
 Non-Homologous End Joining – re-joins cut using Ku protein
and DNA Ligase for deletions, insertions facilitated by DNA with
suitable overhangs
 Homologous Directed Repair – allows DNA sequence to be
incorporated via displacement as long as both sequences have
long homologous elements
 CRISPRi – uses a ‘dead’ form of Cas9 unable to cut DNA,
repressing transcription through methylation
 Dead CRISPR-Cas9 associated with activation-induced cytidine
deaminase, to cleave nitrogenous components of cytosine to
uracil (C:G to U:A)
 In RNA, the TadA enzyme can convert adenine to inosine, which
is functionally similar to guanine
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Human Editing : Contentions
 Germline editing could eradicate inherited
genetic diseases such as β-thalassaemia,
preventing a child being born and suffering with
the condition
 Somatic editing could be used to treat severe
conditions like immunodeficiencies, without
having to worry about transmission to offspring
 Preferential phenotypes are often dependent on
multiple genes, have mechanics that are not yet
known, and too difficult to engineer
Cons
 CRISPR-Cas9 may introduce unexpected off-
target mutations, causing more issues than
initially hypothesised
 Designer babies could be potentially created
by inserting preferential genes (i.e.
intelligence, beauty etc.), pushing eugenics
 Religious arguments contest the idea of
‘playing God’ by interfering with nature
 Could bring legal disputes, gene modifying
techniques are often patented, leading to
arguments about ownership
 Germline-edited offspring have not
consented to the modifications made to their
DNA
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Pros

Medical Applications : Organ Transplantation
 Shortage for human organ donation means that other mammals are often
looked at for alternatives (e.g. Baby Fae with baboon heart for hypoplastic
heart syndrome)
 Rejection caused by foreign antibodies stimulating immune system to
produce xenoreactive natural antibodies or T-cells
 Porcine-derived organs have a low disease transmission, are anatomically
comparable and easily sourced
 Xenozoonosis – transmission of animal infections to humans is a risk as
implantation bypasses physical barrier
 Porcine endogenous retroviruses shown to infect human cell lines in-vitro,
showing a risk of possible reactivation, deactivated all 62 using CRISPR
 Chimeras produced using gene-knockout in embryos to prevent porcine
pancreas development, replacing them with stem cells, but could
undesirable differentiate elsewhere
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Medical Applications : Recombinant Organisms
 Recombinant organisms have their DNA modified by inserting genetic
information from another organism
 Scientists used human β-pancreas cells to extract the DNA sequence for
insulin using polymerase, inserting it into a bacterial plasmid for mass
production
 Plasmids re-adopted by bacteria through transformation, using calcium
ions and temperature changes to alter membrane permeability, success
detected using antibiotics
 Better than using an animal vector (porcine/bovine), as insulin is already
humanised, preventing infection or rejection
 Pharming process use genes for pharmaceuticals into host plants
(tobacco or potato), cultivating them in bioreactors, secreting products
into growth medium
 Strains of rice modified to contain β-carotene genes (psy and crtl), a
precursor of vitamin A to combat deficiency, but faced controversy
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Medical Applications : iPS Cells
 Induced stem cells have pluripotency restored from most adult
cells, using four ‘Yamanaka’ transcription factors
 Use of viral vectors to deliver transcription factors may express
tumour-creating genes, have been successfully removed post-
procedure
 Has less ethical and legal implications as embryos are not used, due
to sanctity of life arguments
 Can be used to model genetic diseases, such as using spinal
muscular atrophy iPS cells, showing that less motor neurone
differentiation occurred, bypassing limitations in sourcing cells
 2017, iPS cells used to combat age-related macular degeneration
(progressive blindness), by forming retinal epithelial cells
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