Conditional Recruitment to a DNA-Bound CRISPR–Cas Complex Using a Colocalization-Dependent Protein Switch

Abstract

To spatially control biochemical functions at specific sites within a genome, we have engineered a synthetic switch that activates when bound to its DNA target site. The system uses two CRISPR–Cas complexes to colocalize components of a de novo-designed protein switch (Co-LOCKR) to adjacent sites in the genome. Colocalization triggers a conformational change in the switch from an inactive closed state to an active open state with an exposed functional peptide. We prototype the system in yeast and demonstrate that DNA binding triggers activation of the switch, recruitment of a transcription factor, and expression of a downstream reporter gene. This DNA-triggered Co-LOCKR switch provides a platform to engineer sophisticated functions that should only be executed at a specific target site within the genome, with potential applications in a wide range of synthetic systems including epigenetic regulation, imaging, and genetic logic circuits.

Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools

“…This switch could control the biochemical functions of specific sites in the genome of S. cerevisiae. 112 Photogenetics is a method that combines light stimulation and genetic manipulation to regulate cellular processes. 113 Photoregulated transcription can be applied to improve the synthesis of valuable products in engineered S. cerevisiae.…”

“…In addition, some chemical molecules (e.g., β-estradiol and plant growth hormone ABA) or various synthetic switches can be used for precise regulation to achieve CRISPR/Cas-mediated control of translational dynamics. , Recently a colocalization-dependent protein switch, Co-LOCK, has been developed that activates when binding to the DNA target site. This switch could control the biochemical functions of specific sites in the genome of S. cerevisiae …”

Programmable protein circuit design

“…However, instead of reconstituting a split effector protein, they took advantage of an engineered protein activation system called LOCKR in which one protein, termed the "key," can release a second protein domain from autoinhibition by a "cage" domain (Langan et al, 2019). Here, recruitment of the key successfully released the adjacent target peptide sequence from the "cage" domain, allowing it to recruit a transcriptional activating domain, effectively converting sequence sensing to transcriptional activity (Kirkpatrick et al, 2020)(Figure 2J). The modular nature of LOCKR in principle allows arbitrary peptide domains to be autoinhibited and released, and should enable orthogonal recruitment of a diverse set of transcriptional effectors, allowing sequence-specific transcriptional modulation in cells.…”

Synthetic Protein Circuits and Devices Based on Reversible Protein-Protein Interactions: An Overview

“…After the 'key' displaces the 'latch' from the 'cage', functional motifs on the 'latch' could engage the interaction with the target. The same technology was used by Kirkpatrick and co-workers (2020) to co-localize LOCKR (co-LOCKR) in target regions in the genome exploiting two Cas9 complexes which are fused to the 'key' and to the 'cage'-'latch', respectively, and used it to specifically promote the transcription of target genes when the two moduli are bound to the DNA target site [49]. Other examples demonstrated the exploitability of coiled coils design to accomplish transcriptional control [31,50].…”