CRISPR/Cas has been developed for targeted mutagenesis in diverse species, including plants. However, precise genome editing via homology-directed repair (HDR) is inefficient in plants, limiting our ability to make large deletions or insertions in the plant genomes. Prime editing increases the control over the desired editing and allows the precise introduction of all types of mutations, including insertion, deletions, and all possible base conversions, albeit at low efficiencies. Here, we designed a dual prime editing system to generate large deletions and precise insertions of sequences by repairing template complementarity. We coupled dual pegRNA with Cas9 nickase (nCas9) to generate deletions and insertions. In another modality, we used dual pegRNA with wild-type Cas9 to generate double-stranded breaks to improve the editing at the targeted sites. We tested dual pegRNAs to delete the last exon in OsCCD7, delete the microRNA targeted sequence in OsIPA, and insert the T7 promoter in the 3'UTR of OsALS. Our results showed a high frequency of targeted insertion of the T7 promoter sequence in the 3'UTR of OsALS with wtCas9 and nCas9. Sanger sequencing analysis showed partial deletions at the targeted locus. Further improvements in the designs of pegRNAs will increase the precise genome insertions and deletions in plants.

Thesis advisor: Jia Niu / High-Throughput Particle Display Screening of RNA-Protein Interactions RNA-protein binding interactions have essential roles in many biological processes including transcriptional and translational control; thus, it is important to quantify the binding affinities of these biological complexes through functional binding assays. Although conventional binding assays have provided significant insight to these dynamic networks, they generally provide a relatively low throughput for a limited number of samples. To overcome the limitations of these conventional binding assays to study RNA-protein binding interactions, we propose to develop an in vitro, high-throughput particle display-based for RNA aptamer screening of RNA-protein complexes for the subsequent identification and characterization of novel RNA aptamers that influence protein binding. With this technique, we will be able to profile large numbers of binding events based on binding-induced fluorescence-enhancement for a more holistic understanding of the corresponding RNA-protein network. So far, we have confirmed that this particle display-based technique can be used to estimate the binding affinity of the well-characterized MS2-MCP model system, and plan to advance this technique to screen a library of MS2 variants for mutational analysis. Prime Editor-Mediated Programmable Insertion of UAAs into Endogenous Proteins The introduction of unnatural amino acids (UAAs) to endogenous cell surface proteins for site-specific bioconjugation reactions allows for the incorporation of clickable, fluorescent handles in vivo; however, the transient expression of proteins harboring UAAs is limited by its transfection efficiency. Thus, we propose to employ prime editors and tRNA/aminoacyl-tRNA synthetase technologies to introduce an UAA to endogenous proteins for downstream bioconjugation applications. Briefly, we propose to stably incorporate a stop codon into mammalian cells by prime editing which will be confirmed with a reporter system, such that this stop codon can mediate the introduction of an UAA through the associated tRNA/aminoacyl-tRNA synthetase technology. By permanently introducing a bioorthogonal, clickable handle onto an endogenous protein, its cellular signaling and localization patters can be monitored in vivo for further classification of the behaviors of these proteins. So far, we identified a promising fluorescent reporter construct to validate the introduction of a stop codon into the mammalian genome by prime editing. / Thesis (MS) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Det finns idag flera tusen genetiska sjukdomar som inte kan botas med hjälp av dagens läkemedelsbehandlingar. Detta är något forskarna försöker finna en lösning på. Två nya potenta genredigeringsverktyg har utvecklats och tros kunna bota och behandla många av de idag kända genetiska sjukdomarna. Detta är clustered regularly interspaced short palindromic repeats med CRISPR-associerade proteiner, CRISPR/Cas9 och prime editing. Tekniker som utvecklats från det adaptiva immunförsvaret hos prokaryoter. Både CRISPR/Cas9 och prime editing är RNA-guidade system med DNA som mål, de är även möjliga att programmera. Syftet med denna litteratursökning var att: 1) Jämföra teknikerna CRISPR/Cas9 och prime editing, 2) Undersöka vilka idag pågående kliniska prövningar som finns där någon av teknikerna används vid behandling av sjukdom. 3) Undersöka vilka sjukdomstillstånd som tros kunna botas och/eller behandlas med hjälp av någon av teknikerna samt 4) undersöka hur forskare ser på de etiska aspekterna av dessa tekniker. Information har hämtats under arbetets gång, främst från PubMed, Google och clinicaltrials.gov. Det finns idag 16 pågående studier där CRISPR/Cas9 används som behandlingsmetod. För prime editing finns det inga pågående studier. Sjukdomarna som forskarna hoppas kunna behandla med hjälp av metoderna är många, men de har kommit längst i utvecklingen av läkemedel för cancer, blodsjukdomar och ögonsjukdomar. De etiska diskussionerna har varit många och den stora frågan som diskuteras är hur tekniken skall regleras för att inte utnyttjas till sådant som potentiellt kan vara skadligt. Detta är två tekniker med hopp om nya behandlingsmetoder för genetiska sjukdomar, dock är de endast i början av sin utveckling och mer forskning och förfining av metoderna krävs innan de kan tillämpas kliniskt. / Today, there are thousands of genetic diseases that cannot be cured with the help of today's drug treatments. This is something the researchers are trying to find a solution to. Two new potent gene editing tools have been developed and are believed to be able to treat or cure many of today's genetic diseases. These are Clustered regularly interspaced short palindromic repeats with CRISPR-associated proteins, CRISPR/Cas9 and prime editing. Techniques developed from the adaptive immune system of prokaryotes. Both CRISPR/Cas9 and prime editing are RNA-guided DNA-targeted systems that are programmable. The purpose of this literature search was to: 1) compare the CRISPR/Cas9 and prime editing techniques, 2) investigate the current clinical trials in which any of the techniques are used to treat disease. 3) investigate which diseases that are believed to be cured and/or treated by using one of the techniques, and 4) investigare how researchers view the ethical aspects of these techniques. Information was gathered during a period between January to May 2020, mainly from PubMed, Google and clinicaltrials.gov. There are currently 16 ongoing studies using CRISPR/Cas9 as a treatment method. For prime editing there are no ongoing studies. The diseases that the researchers hope to be able to treat using the methods are many, but they have come the farthest in the development of a drug for cancer, blood diseases and eye diseases. There have been many discussions about the ethical side, but the big question being discussed is how the technology should be regulated so that it may not be used to harm instead of treat. These two techniques give hope of new treatment methods of genetic diseases, however, they are in the early stages of their development and more research and refinement of the methods is required before they can be applied clinically.

Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques.  This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students.