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Scharenberg Lab

Scharenberg Lab. Aim 1: Work with Baker/Monnat/Stoddard components to characterize new homing endonuclease scaffolds Monomerized homodimers: mMsoI surface displays and exhibits specific binding. No clear cleavage activity as of yet. mCreI does not surface display well Native homodimers

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Scharenberg Lab

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  1. Scharenberg Lab • Aim 1: Work with Baker/Monnat/Stoddard components to characterize new homing endonuclease scaffolds • Monomerized homodimers: • mMsoI surface displays and exhibits specific binding. No clear cleavage activity as of yet. • mCreI does not surface display well • Native homodimers • I-SceI does not surface display well. We have attempted iterative mutation and generated surface displayable variants, but these are not characterized • I-OnuI: in process • Surface display of Ani homologues living in cytB oxidase genes (many available) (Kyle Jacoby)

  2. Scharenberg Lab • Aim 2: Work with Baker/Monnat/Stoddard components to develop strategies for selection of LHEs with novel recognition specificities • Iterative base-by-base selections • Yupeng, Amanda, Audrey, Ryo • Computational design/Binding-based selections: • able to consistently generate fairly sequence specific binding - attempts to transition a population of binders to specific cleavers are in process (Jordan, Yupeng) • Computational/rational design with cleavage-based selection • Some success in generating diverse populations of proteins that retain cleavage activity (Jordan)

  3. Scharenberg Lab • Aim 3: methods development • Increase sensitivity of cleavage assay (Hoku) • Ligate a fluorophore linked oligo onto yeast following an in vitro cleavage reaction to allow “gain of signal” detection • Works as well as fluorophore loss so far, evaluating whether this can be further improved • Library profiling (Hoku) • Sensitivity an issue - we can detect cleavage of a single rare target in a uniform yeast population, but to date are having trouble detecting cleavage of a single rare target by a rare yeast • Tethering strategies to bias repair towards HR (Jeff) • Can a homing endonuclease be linked to proteins/functional domains that influence DNA repair pathway choice?

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