Massively Parallel High-Order Combinatorial Genetics by CombiGEM in Human Cells

Alan S. L. W ong, Gigi C. G. Choi, Allen A. Cheng, Oliver Purcell, Timothy K. Lu

Synthetic Biology Group, MIT Synthetic Biology Center, Research Laboratory of Electronics, Department of Biological Engineering and Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
The systematic analysis of combinatorial genetic interactions, which play significant roles in regulating complex biological traits, has been limited in the throughput and order of complexity of genetic combinations that can be studied through current methods. To overcome these bottlenecks and accelerate the study of combinatorial genetics in human systems, we created the CombiGEM (Combinatorial Genetics En Masse) technology for
the scalable and easy assembly of barcoded combinatorial genetic perturbation libraries. CombiGEM enables multiplexed quantification of all members in a given combinatorial genetic library by using next-generation sequencing technologies. The genetic elements included in CombiGEM libraries can be arbitrary, including gene expression/knockdown constructs, microRNAs, synthetic-biology circuit components, and programmable genome editing tools. We generated high-coverage combinatorial libraries comprising two-wise and
three-wise barcoded genetic components in a lentiviral delivery system for efficient and stable genomic integration in human cells. We have validated the entire pipeline for complex genotype-to-phenotype mapping, including combinatorial library assembly, library verification, pooled screening assays with barcode sequencing, computational analysis, and hit validation, in a relevant human cell model of disease. More broadly, our work establishes a powerful platform for the high-throughput profiling of multifactorial genetic combinations that regulate a myriad of phenotypes relevant to biomedical, biotechnology, and basic science applications.
This work was supported by the NIH New Innovator Award (DP2 OD008435), the Office of Naval Research, and the Ellison Foundation New Scholar in Aging Award, and the Croucher Foundation.