Harvard College

The General Hospital Corporation

Alejandro Chavez

George M Church

Frank J Poelwijk

Benjamin W Pruitt

Johnny Hao Hu

Max Schubert

Eric Kelsic

Xiaoge Guo

Nan Cher Yeo

Christian Schroeder Kaas

Jing Cheng

Suhani D Vora

Franck Poelwijk

A method of screening is provided including providing a combination of a plurality of proliferating cell types wherein each proliferating cell type has a unique associated barcode within its genome that is different from other proliferating cell types of the plurality and wherein each proliferating cell type includes an exogenous gene that when expressed produces an associated phenotype to the proliferating cell type which alters proliferation of the cell type, introducing a perturbation to one or more of the plurality of proliferating cell types, inducing expression of one or more of the exogenous genes, determining the relative number of unique associated barcodes after a period of proliferation, and comparing the relative number of unique associated barcodes to a control relative number of unique associated barcodes to indicate the effect of the perturbation on the one or more of the plurality of proliferating cell types.

Methods of screening using barcoded libraries

Expressing guide nucleic acids (e.g., gRNA) from the same oligonucleotide that contains donor sequence permits the high efficiency, simultaneous transformation of a population of cells with both substrates. Using oligonucleotide chip array technology, one can construct thousands of oligonucleotides with customized gRNA and donor sequence in a cost effective manner. In combination, one can efficiently modify endogenous and exogenous genes.

Library-scale engineering of metabolic pathways

The present disclosure provides methods and compositions of modulating expression of a target nucleic acid sequence in a cell. The method comprises introducing into the cell a nucleic acid sequence encoding a Cas9 fusion protein and a guide RNA, wherein the Cas9 fusion protein and the guide RNA are expressed and co-localize at a target site and modulate the expression of the target nucleic acid sequence.

Methods of modulating expression of target nucleic acid sequences in a cell

The present disclosure provides methods and compositions of modulating expression of a target nucleic acid in a eukaryotic cell. The methods include providing to the cell a guide RNA complementary to the target nucleic acid sequence, providing to the cell a fusion protein, wherein the fusion protein comprises a nuclease null Cas9 protein and a transcriptional effector domain, wherein the nuclease null Cas9 protein interacts with the guide RNA and binds to the target nucleic acid sequence in a site specific manner and wherein the transcriptional effector domain modulates expression of the target nucleic acid.

Cas9-based transcription modulation systems

CRISPR/Cas Systems are provided where a tuned guide RNA is used to discriminate between two protospacer sequences of same length that differ by one nucleotide.

Cas discrimination using tuned guide RNA

Methods of making mutant Cas9 proteins are described.

Mutant Cas9 proteins

A nucleic acid construct is provided that encodes two or more or a plurality of spacer sequences separated by restriction endonuclease recognition site. A plurality of such nucleic acid sequences are provided as a library for making guide RNAs for use with CRISPR/Cas systems.

Methods of making guide RNA

Methods of making deletion mutants of Cas9 proteins and making chimeric Cas9 proteins are described. The Cas9 N- and C-terminal domains may play critical roles in crRNAitracrRNA binding and/or PAM selectivity. To analyze activity, a series of domain exchange mutants between NM and STI (Cas9) were made, replacing the N and/or C terminus of NM (Cas9) with the homologous region from STI. The chimeric proteins were then tested using the transcriptional reporter assay described herein altering the guideRNA and/or Cas9 specific PAM within the reporter to determine the influence of the domain exchanges on protein specificity. None of the N-terminal domain swaps between NM and STI endowed NM with novel properties. The C-terminal exchange generated a NM-STI hybrid that was capable of interacting with the STI crRNAitracrRNA complex and was further able to suppress a reporter with a STI specific PAM.

Growing community of inventors

New York, NY, United States of America

Alejandro Chavez