The Rockefeller University

The Rockfeller University

Massachusetts Institute of Technology

The Broad Institute, Inc.

Harvard College

Luciano Marraffini

Feng Zhang

Patrick D Hsu

Le Cong

David Bikard

Fei Ran

Neville Espi Sanjana

David Benjamin Turitz Cox

Randall Jeffrey Platt

Naomi Habib

Poulami Samai

Robert Heller

Wenyan Jiang

Jon McGinn

Josh Modell

Gregory Goldberg

Dominik Paquet

David Olivier Bikard

Shauiliang Lin

Provided are compositions and methods for selectively reducing the amount of antibiotic resistant and/or virulent bacteria in a mixed bacteria population, or for reducing any other type of unwanted bacteria in a mixed bacteria population. The compositions and methods involve targeting bacteria that are differentiated from other members of the population by at least one unique clustered regularly interspaced short palindromic repeats (CRISPR) targeted DNA sequence. The compositions and methods can be readily adapted to target any bacteria or any bacteria plasmid, or both.

Sequence specific antimicrobials

Provided are compositions, methods, and kits for improving CRISPR-based editing of DNA targets by a CRISPR-associated (Cas) enzyme. The improvement is made by combining the Cas enzyme and a CRISPR targeting RNA a heterologous DNA repair enzyme that is at least one of RecBCD, AddAB, or AdnAB. The heterologous DNA repair enzyme may have inactivated nuclease activity. The method can include using a DNA repair template to introduce one or more changes into the edited DNA. Cells that contain components of the improved CRISPR systems are included, as are kits for making such cells.

Enhancement of CRISPR gene editing or target destruction by co-expression of heterologous DNA repair protein

Provided are Cas9 enzymes that have mutations that enhance their properties, relative to un-mutated Cas9. The altered Cas9 enzymes exhibit i) an increased rate of spacer acquisition, or ii) increased cleavage efficiency of targets with NAG PAMs, or a combination of i) and ii). The altered Cas9 enzymes comprise an amino acid substitution of 1473 and K500 in aor similar Cas9 enzyme. Also provided are polynucleotides, including expression vectors that encode the Cas9 enzymes, cells that contain the polynucleotides, and methods of making and using such cells. The disclosure includes tagging, or labelling bacteria, and for enhancing phage acquired immunity in bacteria, such as those used in industrial processes, including the food and beverage industry, such as the dairy industry. The food products are also included.

Compositions and methods using Cas9 with enhanced spacer acquisition function

Provided are compositions and methods for cleaving a DNA sequence in a cell. The methods involve comprising introducing into a cell a recombinant vector containing a clustered regularly interspaced short palindromic repeats (CRISPR) system. The system includes a CRISPR RNA (crRNA) targeted to a DNA sequence in the cell that is operatively linked to a promoter; and CRISPR-associated enzymes (Cas) 10, Cas6, and at least one Csm protein. The Cas 10 cleaves the DNA sequence only during transcription of the DNA sequence that is operatively linked to the promoter. Also provided are recombinant vectors for modifying cells, cells that contain the recombinant vectors and modifications introduced by them, and kits that include the modified vectors.

Compositions and methods for transcription-based CRISPR-Cas DNA editing

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Brooklyn, NY, United States of America

Luciano Marraffini