Browsing by Author "Rivera Longsworth, Gia Francesca"

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    Expanding the Enzymatic Activity of the Programmable Endonuclease Cas9
    (2018-08-09) Rivera Longsworth, Gia Francesca; Wagner, Daniel
    The CRISPR-Cas9 gene editing system has revolutionized our ability to make targeted mutations in a variety of organisms. In zebrafish, we are able to use microinjections of Cas9 protein or mRNA alongside guide RNAs (gRNA) to create targeted insertions and deletions (indels) at a frequency sufficient for recovery of loss of function alleles. Combined with the many practical advantages of working with zebrafish, CRISPR-Cas9 has enabled many labs to study gene function at a lowered cost. However, there are improvements that can be made to facilitate the knock in of sequences into the genome, a process which remains inefficient in zebrafish due to a low frequency of homology directed repair (HDR), a mechanism necessary for knock in. Our aim is to increase the rate of HDR in zebrafish by modifying Cas9 using two approaches. First, we hypothesize that the long latency period of the Cas9-DNA complex taken with the short cell cycle of the early zebrafish embryo is biasing the repair mechanism against HDR, so I have engineered a CL1 degron-tagged Cas9 (DegCas9) to destabilize the Cas9 protein. A second approach to increasing the rate of knock in is to reduce the number of molecules involved in the HDR process by using an RNA template for recombination. We hypothesize that the rate of HDR can be improved if the recombination template remains in proximity to the DSB. To this aim, I have created a reverse-transcriptase Cas9 fusion and modified gRNAs that include the targeting sequence for recombination. Our goal is to create a Cas9 system that efficiently uses an RNA template for recombination. Results indicate that DegCas9 is capable of inducing mutations at a reduced rate, and that the modified gRNAs for the RTCas9 system do not reduce the effectiveness of Cas9 function. Further studies will include analyzing the spectrum of lesions induced by DegCas9 and creating alternative versions of DegCas9 and RTCas9 using different domains. If successful, these Cas9 constructs will expand the toolkit available for genetic engineering in zebrafish.