DEVELOPING AND EXPANDING CRISPR-CAS PLANT GENOME ENGINEERING SYSTEMS

Abstract

In order to advance plant biology and speed up crop breeding, researchers have used genome engineering tools in their research. Genome engineering with CRISPR has revolutionized agriculture by providing an easy, fast, and accessible tool to induce desirable mutations. This thesis works on addressing problems in the application of CRISPR for plant genome engineering. CRISPR systems are adopted from bacterial immune systems and consists of a Cas endonuclease and a guide RNA (or crRNA). Cas variants have different characteristics and exploring natural variants can provide systems with enhanced or new applications. The first aim in this thesis is to demonstrate novel LbCas12a for genome editing in Arabidopsis. To overcome the temperature sensitivity of LbCas12a, a heat treatment regime was developed. In order to expand LbCas12a use beyond genome editing, a transcription repression system was developed and used successfully for multiplexed repression of two homologs of EDS1. Two crRNA processing systems were compared, and results suggest that either can be used successfully in Arabidopsis. The second aim is to improve Cas9 and Cas12a editing outcomes by creating Cas-effector fusions. Cas9 and Cas12a were fused with six different exonucleases and compared at three targets in rice protoplasts. Several Cas-exonuclease fusions resulted in an increase in editing efficiency and the production of larger deletions. The Cas-exonuclease fusions’ editing efficiency differed between Cas9 and Cas12a, along with the deletion profile. Additionally, chromatin modulating peptides were fused to Cas9, which resulted in higher editing efficiency without altering deletion profiles. These engineered Cas proteins can be used to create unique editing outcomes, and paired with an increased editing efficiency, could be used to target difficult-to-edit target sites for gene knockout and cis-regulatory elements for fine-tuning gene expression. In summary, this work explored new Cas variant LbCas12a, developed multiplex gene repression systems, and compared engineered fusion Cas9 and Cas12a proteins for increased editing efficiency and larger deletions. The developed and improved CRISPR systems expand the number of available targets, improve efficiency, demonstrate novel editing outcomes, and enable multiplexed transcriptional regulation in plants.