Medical Genetics Commons^™

The Revolutionary Genome Editor: Crispr-Cas9 Systems, Grace Spade

Senior Honors Theses

Genetic engineering is the modification of an organism's genetic material to alter its traits through adding, deleting, or changing specific genes. CRISPR-Cas9 systems are groundbreaking tools for genetic engineering, in short utilizing a molecule called RNA to guide a protein called Cas9 to a specific location in DNA to add, delete, or replace genes. The history of how the CRISPR-Cas9 systems came into existence, how it was adapted from a natural defense system in bacteria, and its mechanism of action in both are explained. Its applications, both present and future, competing genetic modifiers, advantages and disadvantages, and the ethical dilemmas …

A Cloning-Free Recyclable System For Crispr-Cas9 Mediated Mutant And Reversion Construction In Candida Albicans Clinical Isolates, Amanda K. Vogel

Theses and Dissertations (ETD)

Candida albicans is a ubiquitous opportunistic fungal pathogen and one of the most prevalent causes of fungal diseases worldwide. The reference isolate SC5314 is one of the most widely used strains for both experimental and genetic studies, but it is becoming increasingly evident that genetic diversity in clinical isolates plays an important role in antifungal resistance, virulence, and pathogenicity. These recent discoveries highlight the need for genetic tools that are capable of investigating genes in multiple strain backgrounds. Here we build on the SAT1-flipper method and combine it with CRISPR-Cas9 technology to achieve cloning-free homozygous deletion in a single transformation …

Using Crispr Gene Editing To Prevent Accumulation Of Lipids In Hepatocytes, Erin Young, Cem Kuscu, Christine Watkins, Murat Dogan

Longitudinal Scholar's Project

CRISPR gene editing is a molecular technology that can be used to silence gene expression. In this experiment, genes that are known to play a role in lipid accumulation in hepatocytes were targeted. Specifically, levels of fatty acid transport proteins 2 and 5 (FATP2 & 5) have been shown to be elevated in cases of non-alcoholic fatty liver disease. The goal of this experiment was to reduce expression of these genes by using a dead Cas9 (dCas9) protein with an attached inhibitory domain (KRAB) that acts on the promotor region. When measuring the mRNA expression, it was determined that the …

Current Prospects And Outlook Of Crispr/Cas Technology In Clinical Therapy, James Piper

Theses and Graduate Projects

Gene editing capabilities have expanded enormously since researchers first demonstrated the robust, accurate, and efficient endonuclease activity of the CRISPR/Cas riboprotein enzyme. Since this gene editing technique was first described in 2013, the prospect of gene therapy as a viable clinical tool has improved immensely. CRISPR/Cas techniques and the methods by which they are delivered into cells, have evolved rapidly since the technology􏰿s inception, and successful, intentional genetic alterations of numerous mammalian cell lines have been reported. As a research tool, CRISPR/Cas9 has already proven itself indispensable in a brief period of time. While there are not numerous clinical trials …

Identification Of Functional Regulatory Elements In The Human Genome Using Pooled Crispr Screens., Samantha M. Borys, Scott T. Younger

Manuscripts, Articles, Book Chapters and Other Papers

BACKGROUND: Genome-scale pooled CRISPR screens are powerful tools for identifying genetic dependencies across varied cellular processes. The vast majority of CRISPR screens reported to date have focused exclusively on the perturbation of protein-coding gene function. However, protein-coding genes comprise < 2% of the sequence space in the human genome leaving a substantial portion of the genome uninterrogated. Noncoding regions of the genome harbor important regulatory elements (e.g. promoters, enhancers, silencers) that influence cellular processes but high-throughput methods for evaluating their essentiality have yet to be established.

RESULTS: Here, we describe a CRISPR-based screening approach that facilitates the functional profiling of thousands of noncoding regulatory elements in parallel. We selected the tumor suppressor p53 as a model system and designed a pooled CRISPR library targeting thousands of p53 binding sites throughout the genome. Following transduction into dCas9-KRAB-expressing cells we identified several regulatory elements that influence cell proliferation. Moreover, …

Hiv Vaccines: Progress, Limitations And A Crispr/Cas9 Vaccine, Omar A. Garcia Martinez

Biology: Student Scholarship & Creative Works

ABSTRACT: The HIV-1 pandemic continues to thrive due to ineffective HIV-1 vaccines. Historically, the world’s most infectious diseases, such as polio and smallpox, have been eradicated or have come close to eradication due to the advent of effective vaccines. Highly active antiretroviral therapy is able to delay the onset of AIDS but can neither rid the body of HIV-1 proviral DNA nor prevent further transmission. A prophylactic vaccine that prevents the various mechanisms HIV-1 has to evade and attack our immune system is needed to end the HIV-1 pandemic. Recent advances in engineered nuclease systems, like the CRISPR/Cas9 system, have …

Insertion Of Sequences At The Original Provirus Integration Site Of Mouse Rosa26 Locus Using The Crispr/Cas9 System., Rolen M. Quadros, Donald W. Harms, Masato Ohtsuka, Channabasavaiah B. Gurumurthy

Journal Articles: Genetics, Cell Biology & Anatomy

Targeted transgenic mouse models, where an exogenous gene is inserted into a specified genomic locus to achieve its stable and reliable expression, have been widely used in biomedical research. However, the available methodologies for targeted insertion of sequences require many laborious steps that involve the use of embryonic stem (ES) cells. We recently developed Pronuclear Injection-based Targeted Transgenesis (PITT), a method that uses a recombinase-mediated cassette exchange (RMCE) to enable insertion of sequences at a predetermined genomic locus, such as ROSA26. The PITT technique uses fertilized eggs (instead of ES cells) collected from 'seed mice' that contain the RMCE landing …