Page 247 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

*[P87] GENE EDITING WITH A TWIST; ENGINEERING CRISPR RESISTANCE INTO TRANSGENIC
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REPORTERS. Magnus Macaulay , Tommy Kuo , Jose Alonso , and Geoffrey Wasteneys . Department
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of Botany, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4; and Department of Plant
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and Microbial Biology, Program in Genetics, North Carolina State University, Raleigh, North Carolina
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Correspondence to: magnus.macaulay@botany.ubc.ca

Gene editing will play a key role in adapting crop plants to increasing climate-associated challenges. One
approach is to introduce a modified version of a gene into a genome. This transgenic approach is
problematic, however, because of competition from the unmodified endogenous gene. This is especially
true for essential, single-copy genes because knocking out an essential gene has lethal consequences. In
this study, we demonstrate a novel approach to generate modified transgenes that are CRISPR-resistant,
so that CRISPR can subsequently knock out only the endogenous copy.

Our essential gene of interest is MICROTUBULE ORGANIZATION 1 (MOR1), which encodes a
microtubule-associated protein that is required for microtubule polymerization in Arabidopsis. Previous
studies have analyzed MOR1 function through conditional mutants since knockout alleles are
homozygous-lethal. The mor1-1 allele, with a L174F single amino acid substitution, causes microtubule
disorganization at temperatures above 29°C, resulting in conspicuous left-handed root twisting. While a
yellow fluorescent protein-tagged reporter of mor1-1 (mor1-1-3xYPet) enables live cell imaging of the
mor1-1 protein, the left-twisting phenotype is masked when the transgene is expressed in either wild-type
or a T-DNA insertion mutant background, indicating that achieving a true null background is difficult. To
overcome this challenge, we first used recombineering technology to modify a MOR1 clone in a
transformable bacterial artificial chromosome. In addition to adding a fluorescent protein-encoding
sequence, and introducing the mor1-1 point mutation, we introduced silent nucleotide substitutions at two
CRISPR protospacer-adjacent motif (PAM) sites near the 5’-end of the MOR1 coding sequence. The
CRISPR-resistant transgenic reporter was then introduced into wild-type Arabidopsis by Agrobacterium-
mediated transfection, and lines homozygous for the transgene were selected in the T2 generation using
antibiotics and fluorescence microscopy. Next, seedlings carrying the CRISPR-resistant mor1-1-3xYPet
were transfected with a CRISPR/Cas9 construct targeting the PAM sites still present in the endogenous
MOR1 gene. Finally, plants with CRISPR/Cas9-induced indels in the endogenous MOR1 gene were
identified by sequencing. Sequencing results confirmed the CRISPR construct only knocked out the
endogenous MOR1. Importantly, we found that expression of the CRISPR-resistant mor1-1-3xYPet
transgene reporter generated the characteristic temperature-dependent left-handed root twisting of mor1-
1 mutants only after CRISPR was introduced.

Our study demonstrates that it is possible to engineer CRISPR-resistant transgenes, an innovative
approach that could have widespread applications for both understanding the function of essential genes
in any organism, and for modifying specific genes in crop species.

[P88] MONOTERPENE INDOLE ALKALOIDS PURIFICATION AND IDENTIFICATION FROM PLANTS
VINCA MINOR AND TABERNAEMONTANA LITORALIS. Zhan Mai and Yang Qu. University of New
Brunswick, department of chemistry, Fredericton, NB, Canada E3B 5A3
Correspondence to: zmai@unb.ca

Monoterpene indole alkaloids (MIAs) are one of the largest classes of alkaloids with diverse bioactivities.
The MIAs are rich in Apocynaceae, Loganiaceae, and Rubiaceae plant families and many MIAs are used
commercially owing to their medicinal values, such as anticancer vinblastine and antimalarial quinine.
To futher understand MIA chemistry and biochemistry, we investigated MIA metabolites in two
Apocynaceae species Vinca minor and Tabernaemontana litoralis. We obtained plant total alkaloids by
standard acid-base extraction from total plant materials. We then used thin Layer Chromatography (TLC)
to efficiently separate and purify MIAs. We used Liquid Chromatography-Mass Spectrometry (LC-MS/MS)
to identify MIA masses and possible skeletons. We further identified major MIAs in these plants by H and
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13 C Nuclear Magnetic Resonance (NMR), and the identification was further supported by comparing to
literature values. 2D-NMR analyses included Correlation Spectroscopy (COSY), Heteronuclear Multiple
Bond Correlation (HMBC), Heteronuclear Single Quantum Correlation (HSQC), and Nuclear Overhauser
Effect Spectroscopy (NOSEY).

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