Genome editing with CRISPR-Cas9 in the Illinois long term selection experiment

Recent advances in genome editing by Clustered Regularly Interspaced Palindromic Repeat (CRISPR) and CRISPR Associated Protein 9 (Cas9) have markedly increased our ability to characterize genes and use genetics to the benefit of agriculture. In this work, we utilize this technology to study the Illinois Long Term Selection Experiment (ILTSE), a unique germplasm resource for studies of genome evolution and genetic variants that contribute to phenotypic traits. ILTSE genotypes, Illinois High Protein 1 and Illinois Low Protein 1, create highly regenerable embryogenic type I callus, enabling transformation and genome editing approaches to characterize gene function. The Lemon White 1 (Lw1) locus was initially targeted as a proof of concept to generate albino plants easily detectable in a population of regenerated plants. Four guide RNAs were tested for their function using an in vitro Cas9 cleavage assay. CRISPR editing vectors were delivered to embryogenic calli using biolistics and transgenic events selected. Multiple albino plants indicative of biallelic mutations were recovered in the ILP1 genotypes at 1.5% efficiency; however none were produced from the IHP1 genotype. A second CRISPR experiment targeted the L-Asparaginase (ASNase) gene, which exhibits reduced gene expression in IHP1 compared to ILP1. The goal was to test whether reducing ASNase gene function can increase grain protein concentration in the ILP1 background. Four guide RNAs were designed and tested in vitro before delivery. Two ILP1 events were generated with novel ASNase deletion alleles. Limited T1 seed was recovered and will be used for future characterization.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Crop Sciences

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2019

Author dc:creator

• Jinga, Stephen Joseph

Contributors dc:contributor

• Moose, Stephen P
• Hind, Sarah
• Jamann, Tiffany

• Maize
• Genome Editing
• CRISPR
• Cas9
• Csy4
• Illinois Long Term Selection Experiment
• ILTSE
• Lemon White 1
• Asparaginase

Statement dc:rights

• Copyright 2019 Stephen Jinga

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/105835

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/105835