CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

Jianting Zhou,Zhongjun Qin,Xiaoli Xue,Ronghai Wu

doi:10.1093/nar/gkw475

Abstract

Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated ‘CasHRA (Cas9-facilitated Homologous Recombination Assembly)’ that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions.

Highlights

The assembly of small DNA fragments into large constructs, such as the genes involved in biochemical pathways, biological machinery, and even entire genomes, is one of the most fundamental requirements of synthetic biology
CasHRA was developed to facilitate the direct use of large circular DNAs for one-step assemblies in vivo and avoid the substantial manipulation of linear DNA segments in vitro
Conventional DNA assembly methods produce purified linear subassembly DNAs by complex manipulations in vitro, which can be burdensome in the last stage of megabasesized DNA assembly

Summary

Introduction

The assembly of small DNA fragments into large constructs, such as the genes involved in biochemical pathways, biological machinery, and even entire genomes, is one of the most fundamental requirements of synthetic biology. The first heavily edited yeast chromosome, synIII (273 kb), was synthesized by 11 successive rounds of DNA assembly in vivo and used to replace the native chromosome III [12]. This method avoided the direct assembly of large DNAs and allowed for stepby-step testing of the functionality of the newly synthesized regions. The assembly efficiency decreases drastically down to 1/48 at the final stage of genome assembly because this method requires a sufficient quantity of high-quality large subassembly DNA segments from complex in vitro manipulation procedures. All eleven 100 kb assembly intermediates were isolated from yeast, and two steps of purification and one step of enrichment were performed to remove the host chromosomal DNA contam-

Methods

Results

Conclusion