High-Throughput Native Context Mapping of the Regulatory Genome

Technology #18012

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Multiplexed editing regulatory assay (MERA). (a) In MERA, a genomically integrated dummy gRNA is replaced with a pooled library of gRNAs through CRISPRCas9–based homologous recombination such that each cell receives a single gRNA. Guide RNAs are tiled across the cis-regulatory regions of a GFP-tagged gene locus, and cells are flow cytometrically sorted according to their GFP expression levels. Deep sequencing on each population is used to identify gRNAs preferentially associated with partial or complete loss of gene expression. (b) Zfp42GFP mESCs show uniformly strong GFP expression. After bulk gRNA integration, a subpopulation of cells lose GFP expression partially or completely. These cells are flow cytometrically isolated for deep sequencing. (c,d) Bulk reads for gRNAs are highly correlated between replicates from the Tdgf1 (c) or Zfp42 libraries (d), indicating consistent and replicable integration rates.
Categories
Inventors
Professor David Gifford
Department of Electrical Engineering & Computer Science, MIT
External Link (cgs.csail.mit.edu)
Professor Richard Sherwood
Computer Science and Artificial Intelligence Laboratory, MIT
Nisha Rajagopal
Computer Science and Artificial Intelligence Laboratory, MIT
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

High-throughput CRISPR-based Library Screening

Provisional Patent Application Filed
Publications
High-throughput mapping of regulatory DNA
Nature Biotechnology, January 25, 2016, pg 167–174

Applications

This invention is a high-throughput CRISPR/Cas9-based approach that analyzes the regulatory genome for function in its native context. Understanding gene regulation for improved control allows for a number of applications including better management of stem cell differentiation.

Problem Addressed

Gene regulation, which underlies human variation, disease, and cancer, is poorly understood making it difficult to predict the effects of cis-regulatory variants on gene expression and to predictively alter gene expression during stem cell differentiation and reprogramming. Currently, there is no high-throughput approach capable of determining the relative importance of each gene regulatory element on native gene expression levels. This technology, Multiplexed Editing Regulatory Assay (MERA), enables the analysis of the regulatory genome at single base resolution in its native context.

Technology

CRISPR/Cas( has been used in genome-wide mutation screens to identify genes required for survival, drug resistance, and tumor metastasis. MERA improves upon this previous gene mutation screening approach by ensuring cells receive a precise number of guide RNA (gRNA) per cell (i.e., one or more than one for combinatorial studies) and allows for gRNA libraries to be used without any laborious molecular cloning into a delivery vector. These methods can be used to screen for the effect of mutations anywhere in the genome.

Advantages

  • Single base resolution
  • No repeated and laborious cloning steps
  • High-throughput