Undergraduate Honors Thesis


Assessing a Viral-Mediated CRISPR/SaCas9 Strategy to Disrupt Local Clocks in Rat Brain Public Deposited

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  • Circadian rhythms, the internal biological processes that regulate physiological and behavioral phenomena in living organisms, are coordinated by the Suprachiasmatic Nucleus (SCN) of the Hypothalamus which fundamentally relies on a complex network of conserved genes, termed “clock genes.” Among these genes, bmal1 (Brain and Muscle Arnt-like protein 1) plays a pivotal role in making up the positive arm of the transcription-translation feedback loop (TTFL), so when bmal1 is knocked out, the clock becomes inoperable. In this project I sought to determine whether there is a time-of-day (TOD) difference in BMAL1 protein expression in the SCN. I also sought to determine whether an intersectional viral strategy could be used to induce genomic knockdown using a Cre dependent CRISPR-SaCas9. Three separate adeno-associated viral (AAV) vectors, containing either Cre recombinase, single-guide RNA (sgRNA) or SaCas9, were co-infused into the SCN. Additionally, we co-infused those same viruses into the Infralimbic Prefrontal cortex (IL-PFC) because of its involvement in fear extinction learning which is under circadian regulation.

    In order to validate BMAL1 protein knockdown, I also developed an immunohistochemistry procedure to measure relative levels of BMAL1 protein. I documented a TOD difference in BMAL1 expression in the SCN with peak expression occurring around ZT1, but only in male rats. Importantly, I found that transduction efficiency within the SCN varied across viral serotypes. Cre, packaged in a retrograde AAV, was localized within the SCN, while the sgRNA and SaCas9, packaged in a different AAV serotype, favored the surrounding area. On top of this, the SaCas9 and sgRNA packaged in the same AAV serotype seemed to optimize expression of both transgenes. Colocalization of the sgRNA and SaCas9 was observed, but further analysis of the extent of BMAL1 knockdown will be required. Because we found good expression of Cre in the SCN, future projects using retrograde AAVs containing the sgRNA and SaCas9 should be explored when trying to get robust transgene expression within the SCN. When targeting regions outside the SCN, the use of similar AAV serotypes should be prioritized when trying to maximize transgene expression. Furthermore, although we experienced difficulties seeing SaCas9 expression, the potential of using the CRISPR-SaCas9 system in a spatial in vivo manner is exciting and should be pursued further.

Date Awarded
  • 2023-11-01
Academic Affiliation
Granting Institution
Last Modified
  • 2023-11-03
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