CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) are two workhorse technologies for loss-of-function studies, yet direct comparisons between the two are scant relative to their widespread adoption. Here, we establish benchmarking libraries for Cas9-based CRISPRko and CRISPRi screens using Perturb-seq as the read-out. For both modalities, we observe consistent transcriptional signatures among cells with the same genes perturbed, strong evidence of on-target signal. We also examine tradeoffs between modalities: while CRISPRi guides demonstrate heightened rates of off-target activity, we also observe artifacts stemming from the cellular response to double-stranded breaks with the use of CRISPRko. The libraries and analyses presented here will be a useful benchmarking and de-risking resource for any group preparing for a large-scale Perturb-seq screen.
This work compares the performance of multiple KRAB domain systems, develops an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed-sequence patterns.
Smriti Srikanth, Fengyi Zheng, Laura M Drepanos et al.· Cell Genomics· 0 citations
While CRISPRi allows direct assessment of target gene repression, CRISPRko provides an effective complementary approach for complete loss-of-function studies, expanding the toolkit for single-cell functional genomics.
Nikhil Gupta, Andrew Sayer, Malwina Prater et al.· bioRxiv· 0 citations
This chapter outlines a comprehensive methodology for the design, assembly, and functional assessment of CRISPR/dCas9 systems optimized for tomato to investigate pathogen-associated responses.
Ananya Mukherjee, Shrabani Basak, Raghuvir Singh et al.· Methods in molecular biology· 0 citations
CRISPR interference (CRISPRi) enables programmable and reversible gene repression but often suffers from leakiness in the uninduced state, thereby confounding phenotypes of essential or dosage-sensitive genes. Here, we introduce a novel CRISPRi architecture, in which dCas9 restricts its own expression through a feedbac...
Perturb-Audit, a diagnostic and denoising framework that integrates molecule-level collision auditing with statistical background suppression, supports an audit-first strategy to improve assignment fidelity and biological interpretability in single-cell CRISPR screens.
Significance Understanding how genes regulate one another is fundamental to biology, yet existing methods for single-cell CRISPR (sc-CRISPR) screens rely on association-based analyses that cannot establish causality and are prone to high false positive rates. We present Canon, an instrumental variable (IV)-based causal...
Peijun Wu, Xiang Zhou· Proceedings of the National...· 0 citations
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