CRISPR Technology Development: Gene Drives and Genome Editing
Date
2020
Authors
Pfitzner, Chandran
Editors
Advisors
Thomas, Paul
Cassey, Phill
Cassey, Phill
Journal Title
Journal ISSN
Volume Title
Type:
Thesis
Citation
Statement of Responsibility
Conference Name
Abstract
The broad theme of this thesis is the development of CRISPR-based genetic engineering technology,
primarily focusing on an exploration of mammalian gene drives.
The CRISPR/Cas9 system utilises a complex composed of the Cas9 nuclease for DNA cleavage and a
guide RNA (gRNA) for targeting it to a specific genomic locus. Since its revolutionary discovery and
utilisation as a genome editing tool, one pioneering application is the CRISPR-based gene drive: The
insertion of the genes for both Cas9 and the gRNA into a specific chromosome in an animal such that
the gRNA targets the homologous locus of the wild type (WT) chromosome. In the offspring of a
cross with a WT animal, the gene drive is initially hemizygous. Subsequently the nuclease and gRNA
complex together and cleave the WT chromosome, resulting in copying of the nuclease and gRNA
genes into that WT chromosome via homology-directed repair (HDR), termed “homing” in the
context of gene drives. When viewed at a population level, this results in the rapid spread of the
gene drive throughout a wild population.
Due to this “Super-Mendelian” inheritance, a gene drive offers the potential to modify entire wild
populations. This opens numerous possibilities such as the eradication or suppression of populations
of invasive pests or immunising natural populations against human pathogens such as malaria in
mosquitoes. These are extremely powerful outcomes that could reduce human disease burden,
reverse the devastating impact of invasive pests on ecosystems, or greatly reduce the agricultural
cost of dealing with pests.
Gene drives have been experimentally tested in a small number of species including the fly
Drosophila melanogaster, the yeast Saccharomyces cerevisiae, and the three mosquito species
Anopheles stephensi, Anopheles gambiae, and Aedes aegypti. All of these have had a very high
homing rate. A low rate of homing has also been observed in Mus musculus in the female germline
but otherwise no vertebrates have experimentally developed gene drives.
This thesis describes the generation of four experimental gene drive approaches in mice, two of
which used Cas9 as the nuclease under the control of either zygotic (CAG) or germline (Vasa)
promoters, and another two that used Cas12a with either zygotic (CMV) or germline (Vasa)
promoters. Gene drives were constructed with the key safety features of a “split drive” and a
“synthetic target” to avoid any ecological impact in case of accidental release.
Homing did not occur at any detectable rate in any of the gene drives. Both the Cas9 zygotic-homing
gene drive and the germline-homing gene drive in males showed a high percentage of indels at the
synthetic target, indicating a high rate of Cas9-induced cleavage. It was concluded that zygotichoming likely failed to occur due to lack of proximity between the gene drive chromosome and the
synthetic target chromosome, as they remain separated a full 18-20 hours post-fertilisation until
after the first G2 phase.
Germline-homing likely didn’t occur in the males as Vasa-induced expression begins during a period
of mitotic proliferation of the primordial germ cells, a cellular state that likely doesn’t promote the
HDR required for homing. Contrasting this, the female oocytes are undergoing meiosis at this time
point, where the homologous chromosomes are aligned and in an ideal position to promote HDR.
However, Cas9 expression levels in the female germline were very low and likely reduced the
chances of any homing occurring.
The Cas12a gene drives all failed to generate an appreciable level of Cas12a cleavage (0-4.3% across
all Cas12a lines), as evidenced by expression levels and percentage of indels observed. As such, the mouse models used to test the Cas12a gene drive here were not sufficient to accurately assess its
functionality.
This thesis also discusses the design and testing of a suite of all-in-one CRISPR gene editing plasmids
that allowed one-step generation of said plasmids containing two unique, customisable gRNAs.
These were all successfully made and showed consistent, simultaneous cleavage of multiple target
sites within cell culture, allowing for multiple knockdowns, large deletions, or reduction of off-target
cleavage via the use of the Nickase variant of Cas9.
School/Discipline
School of Biological Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2020
Provenance
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