Graduation Year

2020

Date of Submission

5-2020

Document Type

Campus Only Senior Thesis

Degree Name

Bachelor of Arts

Department

Biology

Reader 1

Kiana Aran

Reader 2

Emily Wiley

Abstract

Cas12a, a CRISPR protein variant, can be programmed to bind to specific DNA sequences. This binding activates Cas12a’s non-discriminatory single stranded DNase activity (Chen et al., 2018). This has been adapted in a biotechnology setting through diagnostic techniques such as DETECTR (Chen et al., 2018) and HOLMES (Li et al., 2018). Although these methods are quick and low in cost, further work can be done to improve the sensitivity of Cas12a diagnostic methods. Here, we aim to create an in vitro Cas12a diagnostic method through monitoring the degradation of a DNA hydrogel linked to a graphene-based biosensor system. In this research, we use Duchenne muscular dystrophy (DMD) as a proof-of-concept disease. Specifically, this report focuses on the validation of Cas12a’s single stranded DNase activity after its programmed gRNA binds to a target DNA sequence. We programmed Cas12a to target exon 51 of the dystrophin gene, which is deleted in patients with DMD and thus acts as a marker for disease. Subsequent cleavage was monitored using single stranded DNA probes. We expected non-discriminatory cleavage to occur after Cas12a was exposed to healthy DNA and did not expect to see this activity in DMD DNA. However, difficulties in working with the single stranded probes caused our results to be inconclusive. Further work needs to be done to verify Cas12a’s collateral cleavage upon target sequence recognition before proceeding with this diagnostic method.

Download

This thesis is restricted to the Claremont Colleges current faculty, students, and staff.

Share

COinS