Date of Graduation

7-2021

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Srivastava, Vibha

Committee Member

Pereira, Andy

Second Committee Member

Nelson, Christopher E.

Keywords

Crop biotechnology; Genome editing; Genome engineering; Rice

Abstract

Constitutive expression of Cas9 leads to a higher editing efficiency; however, it also increases the chances of off-target mutations. Thus, transient expression of Cas9 is a desirable approach to achieve higher targeting efficiency and to curb the off-target effects. It was previously shown that heat-inducible expression of Cas9 had an editing efficiency of 45% as compared to the strong constitutive expression, and the heat-shock induced mutations were inherited by the next generation. In this study, cold-inducible promoter, AtRD29a, was used for driving Cas9 expression, and evaluated for its editing efficiency upon cold-treatment on the GUS transgene loci. The expression analysis of Cas9 before and after cold-shock showed ~ 2.5x increase in the expression, which is ~1000x lower as that of constitutive expression of Cas9 by rice ubiquitin-1 promoter (OsUbi1). Further, the targeting efficiency of Cas9 expressed under constitutive (OsUbi1:Cas9), cold-shock (AtRD29a:Cas9) or heat-shock (HSP:Cas9) promoters was tested on rice Target of Rapamycin (OsTOR) gene. The OsTOR gene is known to regulate various anabolic processes such as cell cycle, ribosome biogenesis, and photosynthesis. The HEAT repeat regions and kinase domain region of OsTOR were targeted. Among 21 primary transgenic (T0) plants of OsUbi1:Cas9 representing 9 independent transgenic lines, HEAT site was found to be wild type (WT), while monoallelic or biallelic mutations were observed in the Kinase site in 4 plants representing 3 independent lines. The analysis of 33 T1 progeny studied from three T0 plants showed that 22 (66%) inherited the mutations at Kinase domain; but no de-novo mutations in HEAT site were observed. In HSP:Cas9 and AtRD29a:Cas9 lines, none of the 40 T0 plants, which represented 17 independent lines exhibited mutations in either site. The expression analysis of a subset of these lines, showed 2-11x and 2-43x induced transcript levels of Cas9 in HSP:Cas9 and AtRD29a:Cas9 lines, respectively, indicating proper regulation of the Cas9. Beside OsTOR gene, the targeting efficiency of AtRD29a-, HSP- and OsUbi1-CRISPR/Cas9 was tested on the OsPDS gene, the key enzyme in carotenoid biosynthesis pathway. The analysis of Cas9 positive samples in these lines showed only WT sequences in all plants except for one OsUbi1:Cas9, which harbored biallelic mutations, indicating overall lower targeting efficiency in this experiment. In summary, the expression of Cas9 under heat and cold inducible promoters was under proper regulation as it did not exhibit any mutations at room-temperature. However, more analysis is needed to determine whether cold-induced expression of Cas9 by AtRD29a promoter is sufficient to create targeted mutations in the rice genome.

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