# Megabase Scale Genome Engineering and Synthesis in Mammalian Cells

> **NIH NIH DP2** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2020 · $2,431,000

## Abstract

Megabase Scale Genome Engineering and Synthesis in Mammalian Cells
Project Summary:
The design and synthesis of entire genomes provide a powerful strategy for understanding biology and
developing medical applications. However, there is currently limited capacity to perform genome modifications
at very large scale in general, and especially in mammalian systems. Despite limited successes in yeast and
bacteria (Saccharomyces cerevisiae, Mycoplasma, Escherichia coli), the current state of the art in the field of
genome synthesis faces three major challenges: i) how to join synthetic DNA fragments into a very large
contiguous assembly, ii) how to deliver the very large synthetic assembly into the host cell, and iii) how to
integrate the delivered synthetic DNA into defined genomic locations at very large scale. In addition, both i) the
“synthetic minichunk” based integration strategy developed for chromosome synthesis in S. cerevisiae, and ii)
the “genome transplantation” method to replace the entire genome of Mycoplasma species in a single step,
depend on special properties of the host organisms, and thus are unlikely to work in mammalian systems. As a
result, the current progress in large-scale mammalian genome modifications is limited to two examples of locus-
replacements.
Building on my previous success in the total synthesis of a 4-mb recoded E. coli genome, the current pinnacle
across all systems, my newly established lab at Caltech aims to take the next step by expanding such state-of-
the-art genome-writing capacities into mammalian systems. I propose to develop next-generation technologies
to overcome all three challenges aforementioned by enabling: i) megabase assembly of synthetic DNA in
bacteria designed for mammalian expression and subsequent delivery, ii) megabase delivery of synthetic DNA
into mammalian host cells via engineered endosymbiotic bacteria, iii) megabase integration of delivered
synthetic DNA into defined chromosomal locations with high theoretical size capacity and efficiency.
The three aspects of this work (megabase assembly, delivery, and integration of synthetic DNA) are all crucial
for the field of genome synthesis. Combined together, this work aims to establish megabase or larger scale
genome-writing capacities in mammalian systems, and to pave the foundation for the de novo synthesis of an
entire mammalian chromosome and ultimately the entire genome. Once achieved, such revolutionary platform
technologies can significantly accelerate or even transform a wide range of biological and biomedical areas
including – but not limited to – gene therapy, CAR T-cell immunotherapy, humanized animal models, humanized
antibody development, xenotransplantation, and epigenetic studies.

## Key facts

- **NIH application ID:** 10001913
- **Project number:** 1DP2GM140937-01
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Kaihang Wang
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $2,431,000
- **Award type:** 1
- **Project period:** 2020-09-30 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10001913

## Citation

> US National Institutes of Health, RePORTER application 10001913, Megabase Scale Genome Engineering and Synthesis in Mammalian Cells (1DP2GM140937-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10001913. Licensed CC0.

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