Lukasiak, S. | Search bioRxiv

bioRxiv2026

Scarless conditional sgRNAs via endogenous mascRNA processing enable rapid and temporally controlled genome editing

Precise temporal control of gene editing is essential for studying dynamic biological processes, interrogating essential gene function, and improving the interpretability of pooled perturbation screens. Cre-dependent single guide RNA (sgRNA) switches provide temporal regulation by coupling guide activation to site-specific recombination, but existing designs retain a loxP-derived 5' sequence (scar) on the mature sgRNA that can impair guide function. We developed a scarless conditional sgRNA platform that combines Cre-loxP recombination with endogenous RNA processing to restore the native sgRNA architecture following induction. A MALAT1-associated small cytoplasmic RNA (mascRNA) module was positioned upstream of the guide sequence such that, after Cre-mediated recombination, cellular RNase P and RNase Z remove the residual loxP-derived overhang, generating a mature sgRNA with an authentic 5' terminus. Using guides targeting endogenous cell-surface marker genes, the scarless design maintained stringent OFF-state control while improving ON-state editing performance compared with a conventional Cre-activated sgRNA switch, resulting in faster editing kinetics, greater perturbation penetrance, and more consistent editing efficiency. This modular strategy provides a simple approach for conditional CRISPR genome editing that preserves guide integrity and should be readily adaptable to time-resolved functional genomics and pooled screening applications.

cell biology↗

bioRxiv2024

A benchmark comparison of CRISPRn guide-RNA design algorithms and generation of small single and dual-targeting libraries to boost screening efficiency

Genome-wide CRISPR sgRNA libraries have emerged as transformative tools to systematically probe gene function. While these libraries have been iterated over time to be more efficient, their large size limits their use in some applications. Here, we benchmarked publicly available genome-wide single-targeting sgRNA libraries and evaluated dual targeting as a strategy for pooled CRISPR loss-of-function screens. We leveraged this data to design two minimal genome-wide human CRISPR-Cas9 libraries that are 50% smaller than other libraries and that preserve specificity and sensitivity, thus enabling broader deployment at scale.

molecular biology↗