Functional genomics of lipid metabolism in the oleaginous yeast<i>Rhodosporidium toruloides</i>

Coradetti, Samuel T.; Pinel, Dominic; Geiselman, Gina M.; Ito, Masakazu; Mondo, Stephen J.; Reilly, Morgann C.; Cheng, Yu-Ting; Bauer, Štefan; Grigoriev, Igor V.; Gladden, John M.; Simmons, Blake A.; Brem, Rachel B.; Arkin, Adam P.; Skerker, Jeffrey M.

doi:10.1101/190546

Cited by 1 publication

(1 citation statement)

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“…This method was modified from previous Tnseq protocols (Coradetti et al, 2018). Briefly, the transposed plasmids were sheared to $300bp using a S220 Focused-ultrasonicator (Covaris) and purified in between each of the following steps using Agencourt AMPure XP beads (Beckman Coulter).…”

Section: Deep Sequencing Library Preparationmentioning

confidence: 99%

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

Oakes

Fellmann

Rishi

et al. 2019

Cell

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The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.

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Section: Deep Sequencing Library Preparationmentioning

confidence: 99%