MULTIPLEX TARGETED MUTATION AND ANALYSIS OF RICE MAP KINASE GENES WITH CRISPR/CAS9
Open Access
- Author:
- Minkenberg, Bastian
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 27, 2017
- Committee Members:
- Yinong Yang, Dissertation Advisor/Co-Advisor
Yinong Yang, Committee Chair/Co-Chair
David R. Huff, Committee Member
Claude dePamphilis, Committee Member
Gabriele Monshausen, Outside Member - Keywords:
- genome-editing
CRISPR/Cas9
rice
Oryza sativa
off-target prediction
mitogen-activated protein kinases
essential genes
multi-gene family
multiplex - Abstract:
- Traditionally, it was very cumbersome to achieve specific changes in the genome of an organism. Recent advances in site-specific nucleases revived the field of genome-editing and promise to treat diseases and to improve crops. The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system provided researchers with an easy to handle and effective tool for genome-editing. Cas9 can be reprogrammed for a new target by changing a 20 nt sequence in the guide RNA (gRNA). This same feature also allows creation of a flexible multiplex genome-editing tool when several gRNAs are simultaneously provided. However, robust and efficient expression of multiple gRNAs remained a challenge. To overcome this problem, our laboratory developed a robust and efficient method to produce multiple gRNAs from a single transcript expressed by a polycistronic tRNA-gRNA gene (PTG). Chapter 2 of my dissertation describes successful adaptation of PTGs to create stable transgenic rice lines with up to eight mutated target sites and up to 86% mutation frequency. Comparing the PTG/Cas9 system with the traditional CRISPR/Cas9 system revealed that use of PTGs can boost mutation frequency in stable lines from 40-60% to fully 100% in two studied cases. The ability to mutate several genes at once facilitates the functional analysis of redundant genes and multi-gene families. The phenotypes of single knock-out mutants can be masked by redundant functions shared between closely related genes and creating multiple-tuple mutant is necessary to uncover gene functions. Researcher previously needed to cross mutant lines in a time-consuming process. PTG/Cas9, on the other hand, allows for creation of several knock-out alleles in a single transformation. I took advantage of PTG/Cas9’s ability to mutate several genes in chapter 3 of my dissertation to create a mutant library of closely related and stress-responsive rice MPK genes. In this study, I demonstrated 45 to 86% frequency of biallelic mutations and successful creation of single, double and quadruple gene mutants by simultaneously targeting two to eight genomic sites in four MPK genes. I started the analysis of single MPK mutants and identified MPK1 and MPK6 as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR-edited mutants. The true knock-out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, functional importance of specific MPK features could be evaluated by characterizing CRISPR-induced allelic variation in the conserved kinase domain of MPK6. Indels and fragment deletion were both stably inherited to the next generations and transgene-free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. While working on genome-editing with CRISPR/Cas9 during my dissertation research, I discovered that most available online tools fail to predict important off-target sites. I therefore conducted a study described in chapter 4 that aimed to find the reasons for their underperformance. I discovered that all off-target prediction tools I tested were imprecisely developed because they did not adjust their strategy and options for short sequences such as the 20 nt spacer sequences used to guide Cas9. The tested tools especially struggled with similar sequences that required small word-sizes to be aligned to the query and sequences with gaps compared to the query sequence. I developed a new strategy to improve off-target prediction based on sequence similarity by combining global and local alignments to allow for better gap detection and adjusting the options of the used aligners to increase sensitivity. Implementation of this new strategy yielded lists of highly specific CRISPR/Cas9 target sites for seven model and crop plant species. Depending on the species, between 65.4% and 92.6% of the coding sequences in a given genome can be specifically targeted by the spacers provided. These lists will be used to update the CRISPR-PLANT website so that researchers around the world can benefit from these high-stringent target sites in their experiments. My efforts will hopefully minimize some of the current concerns about off-target effect of CRISPR/Cas9 genome-editing.