Crispr cas9 phd thesis

PhD thesis, University of Trento. However, the in vivo application of this technology is still severely limited by unwanted genomic cleavages, that are further increased by long-term expression of the nuclease and can lead to unpredictable results. To address this limitation, we developed a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets in order to select optimized Streptococcus pyogenes Cas9 SpCas9 variants. Through the combination of the identified mutations within a single variant we isolated the best performing nuclease, that we named evoCas9 evolved Cas9. Side by side analyses with recently reported rationally designed variants demonstrated a significant improvement in fidelity of our evoCas9. This self-limiting circuit, by controlling Cas9 levels, results in increased genome editing specificity.
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PhD Dissertation Defense - Pranam Chatterjee

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PhD Dissertation Defense - Pranam Chatterjee — MIT Media Lab

Explore more content. Cite Download Targeted manipulation of the mammalian genome has been revolutionized by the RNA-guided nuclease, Cas9, from the S. A biologist can now target virtually any region of the genome to induce mutations, activate or repress transcription, and compact or decompact chromatin. Cas9 activities can be highly multiplexed within a single cell, providing an unprecedented opportunity to manipulate the mammalian genome and to program cellular behaviors.
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CRISPR Cas9: From Yogurt to Human Genome

Robert Heler. Clustered regularly interspaced short palindromic repeat CRISPR loci and their associated Cas proteins provide adaptive immunity against viral attack in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets protospacers.
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A CRISPR-Cas9 -based gene editing therapy promoted the production of a smaller but functional version of the dystrophin protein in a new mixed mouse model that uses muscle cells derived from Duchenne muscular dystrophy DMD patients. This is one of the first studies to prove the efficacy of this type of gene editing therapy in adult cells within a living organism, as opposed to patient cells grown in a lab dish. According to researchers, this type of data is important to support the evaluation of this gene-editing strategy in patients.
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