CRISPR-based shuttle cloning of 1397 human genes into UAS vectors

Abstract

Functional genomics is a powerful tool for elucidating the function of all genes in an organism and primarily relies on construction and manipulation of genome-wide DNA libraries such as cDNAs, ORFs, gene promoters and inverted repeats for RNAi. Typically, a DNA library must be transferred to a destination vector in a high-throughput manner for functional genomics studies. However, the construction of genome-wide DNA libraries, such as cDNA/ORF overexpression libraries, has been challenging due to limitations in high-throughput DNA cloning methods. This severely restricts the use of functional genomics. The prevailing high-throughput cloning methods for constructing and manipulating genome-wide DNA libraries primarily include Gateway, In-Fusion, Creator, and Univector cloning systems, all of which are based on site-specific recombination. Among these, the Gateway cloning system is the most extensively employed high-throughput cloning method. All the aforementioned high-throughput cloning methods predominantly rely on PCR amplification of the DNA fragments of interest. This step requires individualized manipulations for each DNA fragment, including primer design and synthesis, gel purification, and DNA sequencing, which are laborious and time-consuming. Additionally, PCR amplification is particularly problematic for long DNA fragments. Consequently, the PCR amplification of DNA fragments of interest is not only costly but also a rate-limiting step in high-throughput cloning. For example, although cDNA and ORF resources for human, mouse and Drosophila have been publicly available for nearly two decades, the construction of a genome-wide GAL4/UAS (upstream activating sequence)-based UAS-cDNA/ORF plasmid library from these resources has been severely impeded by the PCR amplification of cDNAs and ORFs [1]. We previously developed a high-throughput cloning method, CRISPRmass, for constructing a genome-wide UAS-cDNA/ORF plasmid library from publicly available cDNA/ORF resources [2]. However, CRISPRmass is applicable solely to the insertion of an identical DNA fragment (e.g., a UAS module) into the identical backbones of different plasmids [2]. It does not allow for the transfer of DNA fragments (e.g., cDNAs or ORFs) between vectors, thereby limiting its use in DNA cloning. By introducing the concept of a CRISPRshuttle cassette, we developed a novel high-throughput DNA cloning method termed CRISPR-based shuttle cloning (CRISPRshuttle cloning). This method allows for the transfer of numerous DNA fragments of interest from original plasmids with identical backbones to a different vector background through two-step test tube reactions prior to bacterial transformation, thereby eliminating the need for PCR amplification of the DNA fragments (Figure 1A). In the first-step test tube reaction, different DNA fragments of interest are excised from their original plasmids by digesting the plasmid backbones with Cas9/sgRNA 1 and Cas9/sgRNA 2. Cas9/sgRNA 1 targets the backbone sequence adjacent to the 5′ end of the DNA fragments, while Cas9/sgRNA 2 targets the backbone sequence adjacent to the 3′ end. The released DNA fragments do not need to be purified, and the reaction products can be directly used in the second-step test tube reaction. In the second-step test tube reaction, the released DNA fragments are transferred to the CRISPRshuttle cassette of a CRISPRshuttle-compatible destination vector via Gibson assembly, yielding the desired plasmids. A CRISPRshuttle cassette consists of approximately 20‒40 bp of backbone sequence flanking both the 5′ and 3′ ends of the DNA fragments, and one or two unique restriction enzyme recognition sites between these sequences. These recognition sites are used for linearizing the CRISPRshuttle-compatible destination vector and must be unique within the vector.

1. Introduction

Functional genomics is a powerful tool for elucidating the function of all genes in an organism and primarily relies on construction and manipulation of genome-wide DNA libraries such as cDNAs, ORFs, gene promoters and inverted repeats for RNAi. Typically, a DNA library must be transferred to a destination vector in a high-throughput manner for functional genomics studies. However, the construction of genome-wide DNA libraries, such as cDNA/ORF overexpression libraries, has been challenging due to limitations in high-throughput DNA cloning methods. This severely restricts the use of functional genomics.

The prevailing high-throughput cloning methods for constructing and manipulating genome-wide DNA libraries primarily include Gateway, In-Fusion, Creator, and Univector cloning systems, all of which are based on site-specific recombination. Among these, the Gateway cloning system is the most extensively employed high-throughput cloning method. All the aforementioned high-throughput cloning methods predominantly rely on PCR amplification of the DNA fragments of interest. This step requires individualized manipulations for each DNA fragment, including primer design and synthesis, gel purification, and DNA sequencing, which are laborious and time-consuming. Additionally, PCR amplification is particularly problematic for long DNA fragments. Consequently, the PCR amplification of DNA fragments of interest is not only costly but also a rate-limiting step in high-throughput cloning.