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Development of Cas9-expressing plants to study gene function

Chakraborty, Anindita (2020) Development of Cas9-expressing plants to study gene function. PhD thesis, Murdoch University.

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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing system requiring activity of a nucleoprotein complex consisting of Cas9 nuclease and a single guide RNA (sgRNA). Within the nucleus of the cell, pairing of the sgRNA with the complementary region of the genome guides Cas9 to cleave the dsDNA molecule at a site located three bases upstream of a protospacer adjacent motif (PAM). Inaccurate repair of cleavage potentially results in insertion/deletion mutations (indels) leading to frame-shifts. This system enables a relatively precise mutation of genes.

The CRISPR/Cas9 system is usually introduced to plants through stable introduction of a cassette consisting of a Cas9 gene, a selectable marker gene, and one or more sgRNAs targeted towards one or specific sites. The disadvantage of this system is that a new transgenic plant must be developed for each new target(s). This study attempted to overcome this limitation. The aim of this project was to develop a homozygous, single-copy, Cas9-expressing Nicotiana tabacum (tobacco) line, and to explore means by which sgRNA molecules could subsequently be delivered to the nucleus of such a plant to affect mutation, leading to phenotypic change.

To achieve the aim, Agrobacterium tumefaciens was used to introduce Cas9 along with an herbicide-tolerance marker gene bialaphos resistance (bar) into tobacco cells. The successful integration of the Cas9-bar cassette in the tobacco genome was confirmed in 43 T0-generation events. Evidence of expression of the Cas9 gene was splicing of its intron upon transcription in plant cells. The gross morphologies of transgenic Cas9-bar-containing plant lines derived from a single event were recorded. Some plants had unusual traits, including bifurcated leaves and fruit, indicative of somaclonal variation had occurred during the tissue culture stage. Single-copy homozygous Cas9-bar-containing lines were identified from segregation patterns of the bar after self-pollination for two sexual generations. One homozygous, single-copy line, named T1-12.2, was selected for further studies.

Three sgRNAs targeting a fragment of the endogenous tobacco plant gene phytoene desaturase (PDS) and five sgRNAs targeting the transgene bar were designed. Cas9-digestion assays were conducted in vitro to confirm the activity of sgRNAs. Cleavage of the target regions was confirmed for two sgRNAs against the PDS gene and two against the bar gene. Seed imbibition, transient expression by agroinfiltration, and stable integration by Agrobacterium-mediated leaf disc transformation methods were used to introduce sgRNAs into T2 or T3 plants from line T1-12.2. Seed imbibition method targeting the bar gene, and agroinfiltration method targeting both the PDS and bar genes failed to generate detectable mutants. Stable transformation of sgRNAs targeting the bar gene facilitated a point mutation, which inactivated tolerance to the herbicide glufosinate. Sequencing confirmed a frame-shift mutation located three nucleotides upstream of a protospacer adjacent motif. Detection of this mutation event provided strong evidence that Cas9 was active in plants of line T1-12.2. Although an efficient delivery method for transiently-expressed sgRNAs was not identified in this project, the T1-12.2 plant line is potentially a valuable tool in which further functional genomics study of genes or its regulatory elements can be done by applying sgRNAs sequentially or simultaneously.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): College of Science, Health, Engineering and Education
Western Australian State Agricultural Biotechnology Centre
Supervisor(s): Wylie, Steve, Li, Hua, Iqbal, Sadia and Jones, Michael
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