CRISPR-based strategies open many new avenues to engineer genomes. CRISPR/Cas9-based mutations are the most commonly used application at the moment. However, a strategy to use CRISPR-Cas9 for the replacement of a genomic DNA sequence with a user defined "donor DNA" sequence at a precise position defined by guide-RNA cleavage ("knock-in" mutation) has recently been proposed to work efficiently in plant genomes (1). Knock-in mutants have many advantages over knock-out mutants, for example protein function can be altered in a precisely defined way that makes it more active.
You will generate several knock-in mutants in Arabidopsis thaliana. We will begin by using the published procedures and compare which have the best efficiencies in our hands. Next, we will consider strategies to optimize the existing methods based our observations. Several potential regions would be of interest to the lab, and the targetes would be chosen in collaboration with the students. Potential examples include: DNA regions giving rise to long non-coding RNA transcripts, knock-in mutations of RNA polymerase II elongation factors, factors affecting the epigenetic architecture of plant genomes and attempts to improve RNA polymerase II transcriptional termination by inerting "terminators" at specific DNA sequences involved in cold-sensing of plants (2).
(1) knock-in strategy: https://www.nature.com/articles/s41467-018-04416-0
(2) Marquardt lab publication: https://www.nature.com/articles/s41467-018-07010-6
Methods used:CRISPR, biochemitry, molecular biology, functional genoomcs, next-generation sequence analysis
Keywords:CRISPR, CAS9, RNA, Chromatin, Epigenetics