Modular Synthesis of sgRNAs and Applications for Gene Editing in Mammalian Cells

• Author / Creator

  Tijaro Bulla, Santiago

• The precise and targeted modification of cell genomes has been an important goal since the discovery of the relationship between DNA sequence and the phenotypes of organism. Many tools have emerged to address this challenge, the most important one being the CRISPR-Cas9 system. This technology consists of a ribonucleoprotein complex made of an endonuclease, called Cas9, and an RNA strand, which is called single guide RNA (sgRNA). Different methods can be used to synthesize these two components, but for the RNA, solid phase synthesis is an approach that allows the precise incorporation of chemically modified nucleotides at any site of the strand. However, this technique is limited by low yields when the desired strand size approaches and exceeds 100 nucleotides. Here, we explore the modular synthesis of a synthetic sgRNA by bringing together three smaller fragments using the copper catalyzed azide-alkyne cycloaddition reaction as a chemical ligation method. This method allows for the incorporation of chemically modified nucleotide into the RNA strand, such as a fluorophore modification that we show allows for the selective enrichment of cells with high concentration of CRISPR reagents through fluorescence activated cell sorting (FACS), increasing the chances of gene editing. We also tested different modifications to increase the stability of the RNA against hydrolysis and found a modification pattern in which the sgRNA could perform gene editing in CHO cell with high yields.
  The simple components of the CRISPR-Cas9 system have allowed it to be used in many applications that expand gene editing. For examples, plasmids with genes encoding both the sgRNA and Cas9 can be delivered to cells though lentiviruses to modify many genes in one single experiment. However, for future clinical application there are advantages to directly introducing the Cas9 and multiple sgRNA into cells. Therefore, we have investigated whether our modular sgRNAs, alongside FACS, could perform multigene editing. We synthesised two sgRNAs targeting Siglec-3 and Siglec-7, which were labelled with different fluorophores. With FACS, we were able to detect and sort and enrich cells that contained the highest concentrations of both sgRNA, which let us to obtain values of double gene knock-out ranging from to 2 to 4%. We also determined a set of conditions in which our sgRNA could be conjugated to a DNA strand where future studies allowed it to be employed as a tool for sequence insertion, or gene knock-in.

• Subjects / Keywords

  • CRISPR
  • sgRNA
  • Modular Synthesis
  • Chemical Modifications
  • Gene Editing
  • Mammalian cells
  • Solid Phase Synthesis

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-06g8-p162

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.