David Colognori

Spligation enables programmable chimeric RNA generation in living cells

David Colognori, Kevin M. Wasko, Marena I. Trinidad, Zehan Zhou et autres

The ability to precisely modify RNA offers opportunities to manipulate the flow of genetic information and influence transcript stability, localization and translation. RNA-targeting technologies enable RNA knockdown, base editing and trans -splicing, but more extensive transcript changes typically require genome editing or …

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Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing

Honglue Shi, Noor Al-Sayyad, Kevin M. Wasko, Marena I. Trinidad et autres

RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with …

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CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d

Benjamin A. Adler, Muntathar J. Al-Shimary, Jaymin R. Patel, Emily G. Armbruster et autres

Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because …

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Single-molecule live-cell RNA imaging with CRISPR–Csm

Chenglong Xia, David Colognori, X. Jiang, Ke Xu et autres

Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule …

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BPS2025 - Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing

Honglue Shi, Noor Al-Sayyad, Kevin M. Wasko, Marena I. Trinidad et autres

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Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing

Honglue Shi, Noor Al-Sayyad, Kevin M. Wasko, Marena I. Trinidad et autres

SUMMARY RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a 20-base-pair DNA sequence adjacent to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical and cell-based assays on S. pyogenes Cas9 ( Spy …

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Single-molecule live-cell RNA imaging with CRISPR-Csm

Chenglong Xia, David Colognori, X. Jiang, Ke Xu et autres

Abstract High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescence in situ …

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CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes

Benjamin A. Adler, Marena I. Trinidad, Daniel Bellieny‐Rabelo, Elaine Zhang et autres

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic …

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Genome-wide Characterization of Diverse Bacteriophages Enabled by RNA-Binding CRISPRi

Benjamin A. Adler, Muntathar J. Al-Shimary, Jaymin R. Patel, Emily G. Armbruster et autres

Abstract Bacteriophages constitute one of the largest sources of unknown gene content in the biosphere. Even for well-studied model phages, robust experimental approaches to identify and study their essential genes remain elusive. We uncover and exploit the conserved vulnerability of the phage …

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CasPEDIA Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes

Benjamin A. Adler, Marena I. Trinidad, Daniel Bellieny‐Rabelo, Elaine Zhang et autres

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic …

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Precise transcript targeting by CRISPR-Csm complexes

David Colognori, Marena I. Trinidad, Jennifer A. Doudna

Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR …

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Decorating chromatin for enhanced genome editing using CRISPR-Cas9

Evelyn Chen, Enrique Lin-Shiao, Marena I. Trinidad, Mohammad Saffari Doost et autres

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative …

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