Harvard College

University of California

Ludwig Institute for Cancer Research Limited

Shape Therapeutics Inc.

Prashant G Mali

George M Church

Luhan Yang

Kevin Michael Esvelt

Kun Zhang

Yan Wu

Dhruva Katrekar

Udit Parekh

Kristen Turner

Reza Kalhor

Viraj Deshpande

Richard Sullivan

Rafael Ponce

Christopher Nye

David Huss

Nathan Palmer

Liana Stein

Anupama Lakshmanan

Doruk Beyter

Susan Byrne

Amir Dailamy

Daniella McDonald

Paul Mischel

Dongxin Zhao

Michael Hu

Ana Moreno Collado

Vineet Bafna

Yiannis Savva

This disclosure relates to methods, polynucleotides, vectors, viral particles, cells, and systems or the engineering of human tissues. One aspect of the disclosure relates to using lineage-specific miRNA binding molecules to bias tissue lineage. Another aspect of the disclosure relates to using lineage-specific transcription factor overexpression to bias tissue lineage.

Method to engineer transplantable human tissues

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

RNA-guided human genome engineering

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

RNA-guided transcriptional regulation

Aspects of the disclosure relate to a gene therapy approach for diseases, disorders, or conditions caused by mutation in the stop codon utilizing modified tRNA. At least 10-15% of all genetic diseases, including muscular dystrophy (e.g. Duchene muscular dystrophy), some cancers, beta thalassemia, Hurler syndrome, and cystic fibrosis, fall into this category. Not to be bound by theory, it is believed that this approach is safer than CRISPR approaches due to minimal off-target effects and the lack of genome level changes.

RNA targeting of mutations via suppressor tRNAs and deaminases

A guide RNA comprising: a gRNA spacer sequence at the 5′ end of the guide RNA, wherein the spacer sequence is complementary to a target gene, a scaffold sequence that binds to Cas9, and an RNA capture and sequencing domain comprising: a barcode sequence, and a primer binding sequence; nucleic acids and vectors encoding the guide RNA; cells expressing the guide RNA; and a library comprising a plurality of guide RNAs. Also disclosed are methods of introducing a genetic perturbation into a cell, methods of assessing an effect of at least one genetic perturbation on RNA expression in a cell, methods of identifying nucleic acid sequences associated with a disease state and a method of identifying candidate therapeutic agents.

Efficient genetic screening method

Understanding the complex effects of genetic perturbations on cellular state and fitness in human pluripotent stem cells (hPSCs) has been challenging using traditional pooled screening techniques which typically rely on unidimensional phenotypic readouts. Here, Applicants use barcoded open reading frame (ORF) overexpression libraries with a coupled single-cell RNA sequencing (scRNA-seq) and fitness screening approach, a technique we call SEUSS (ScalablE fUnctional Screening by Sequencing), to establish a comprehensive assaying platform. Using this system, Applicants perturbed hPSCs with a library of developmentally critical transcription factors (TFs), and assayed the impact of TF overexpression on fitness and transcriptomic cell state across multiple media conditions. Applicants further leveraged the versatility of the ORF library approach to systematically assay mutant gene libraries and also whole gene families. From the transcriptomic responses, Applicants built genetic co-perturbation networks to identify key altered gene modules. Strikingly, we found that KLF4 and SNAI2 have opposing effects on the pluripotency gene module, highlighting the power of this method to characterize the effects of genetic perturbations. From the fitness responses, Applicants identified ETV2 as a driver of reprogramming towards an endothelial-like state.

Methods for screening genetic perturbations

Disclosed herein are compositions that comprise engineered polynucleotides, pharmaceutical compositions comprising the same, methods of making the same, and methods of treatment comprising the compositions that comprise the engineered polynucleotides.

Therapeutic editing

A functional engineered guide RNA sequence is provided including a spacer sequence and a scaffold sequence, wherein the scaffold sequence includes a primer binding site for reverse transcription.

Engineered guide RNA sequences for in situ detection and sequencing

Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

Growing community of inventors

Somerville, MA, United States of America

Prashant G Mali