Producing gRNAs for Use in CRISPR-Cas9 DNA Editing Applications via In Vivo T7 RNA Polymerase Transcription in Escherichia coli.

Author

Christian A. Santiago, Rollins CollegeFollow

Date of Award

Spring 2023

Thesis Type

Open Access

Degree Name

Honors Bachelor of Arts

Department

Biology

Sponsor

Dr. Jay Pieczynski

Committee Member

Dr. Pamela Brannock

Committee Member

Dr. Sabrice Guerrier

Abstract

The development of the CRISPR-Cas9 system is widely considered to be a breakthrough advancement in gene editing. Compared to traditional technologies, CRISPR is generally regarded as the most accessible, effective, and precise tool for inducing stable and inheritable gene edits in vivo. The CRISPR-Cas9 gene editing complex relies on the complementary binding of guide RNAs to specifically target a DNA sequence for endonuclease cleavage and mutagenesis. The production of gRNAs for use in CRISPR applications can become exceedingly complex and costly, as in vivo methodologies generally require the use of RNA polymerases that interfere with gRNA sequence identity and in vitro methodologies rely on chemical synthesis by third party manufacturers. In this study, we present a novel methodology for the in-house synthesis of gRNAs. T7 promoter-gated gRNA-encoding plasmids are transformed into Escherichia coli, expressed via the E. coli’s natively expressed T7 RNA polymerase (which can transcribe RNA without interfering with sequence identity), and processed via RNA purification to isolate transcribed gRNAs. We determined that high quality gRNAs can be produced in high quantities using this method and can furthermore be used to direct in vitro Cas9 DNA digestion. We also attempted to utilize gRNAs produced via this methodology to promote in vivo CRISPR gene edits in Caenorhabditis elegans nematodes with suboptimal results. The results of this study serve to make Cas9-DNA editing applications more widely accessible, as they bear the potential to ease technical and financial barriers of entry that can dissuade scientists from utilizing CRISPR in their research studies.

Comments

Dr. Christopher Fuse also served as a member of the committee.

Recommended Citation

Santiago, Christian A., "Producing gRNAs for Use in CRISPR-Cas9 DNA Editing Applications via In Vivo T7 RNA Polymerase Transcription in Escherichia coli." (2023). Honors Program Theses. 206.
https://scholarship.rollins.edu/honors/206

Rights Holder

Christian A. Santiago