Biology subjects

Kailass, K.

Publications and source records attributed to Kailass, K..

3 recordsLinked to original sources

bioRxiv2026

A Scalable Design for Proximity-Inducing Molecules

Chimeric molecules, which bring together an effector enzyme and a protein-of-interest (POI) to add/remove post-translational modifications (PTMs), are furnishing transformative modalities (e.g., PROTACs). However, these chimeras scalability is limited as they employ rare, non-inhibitory binders of effectors. We report GRoup-transfer chimeras for Inducing Proximity (GRIPs) that employ abundantly available effectors inhibitors to append POI binder on the effector using group-transfer handles. To demonstrate scalability, we develop 6 GRIPs classes for 3 PTMs utilizing diverse inhibitor, spanning 16 effector-POI pairs. Furthermore, we report a toolbox of 42 tunable group-transfer handles for Cys/Lys residues and [~]5000 inhibitor-residue pairs for diverse effectors. Using global proteomics, we confirm the specificity for group transfer and PTM editing. GRIPs endowed new functionalities to POI drugs, including preventing rebound signaling upon drug withdrawal, a more potent/persistent inhibition, and inhibitor-induced pathway activation in 4 fully-endogenous systems. In diverse hemi-endogenous systems (tagged POI), GRIPs induced condensate formation with reduced off-targets, cleared pathogenic PTMs, and initiated PTM crosstalk. Overall, GRIPs provide a scalable and versatile platform for PTM editing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/706349v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@132f70dorg.highwire.dtl.DTLVardef@f9aa62org.highwire.dtl.DTLVardef@12143edorg.highwire.dtl.DTLVardef@f296d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells

Engineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells. SIGNIFICANCE STATEMENTCRISPR technologies are typically engineered in bacteria, even though they must function in the far more complex environment of human cells. This gap has limited the discovery of variants with improved DNA recognition or with resistance to inhibitors that operate differently in mammalian systems. Here we establish CRISPR-MACE, a continuous evolution platform that leverages pressure from anti-CRISPR proteins to select Cas9 variants directly in human cells that have novel functions. Evolved variants show improvements in DNA binding strength and residence time, as well as striking escape from the potent Cas9 inhibitor AcrIIA4. Many anti-CRISPR proteins use distinct mechanisms, so our strategy can drive future continuous evolution campaigns in mammalian cells that expand the functional properties of genome-targeting agents.

synthetic biology↗

bioRxiv2025

Orthogonal resistance mechanisms of classical- and induced-proximity inhibitors

Resistance development is an inevitable failure mode of many drugs, pointing to the need to develop agents with orthogonal resistance mechanisms. Induced-proximity modalities, an emergent class of therapeutics, operate by forming a ternary complex with the protein-of-interest (POI) and effectors, unlike classical inhibitors that form binary complexes with the POI. Using KRAS as a model system, we employed base editor tiling mutagenesis screening to show that induced-proximity inhibitors exhibit orthogonal resistance mechanisms to classical inhibitors despite overlapping binding sites, offering an opportunity to circumvent resistance mechanisms of classical inhibitors. These findings highlight the use of base editor mutagenesis screens to prioritize inhibitors with orthogonal resistance mechanisms and the potential of induced-proximity inhibitors to overcome the drug resistance of classical inhibitors.

biochemistry↗