CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library

Summary

We present a protocol for CRISPR-based modular assembly (CRISPRmass), a method for high-throughput construction of UAS-cDNA/ORF plasmid library in Drosophila using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various plasmid libraries.

Abstract

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5' end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila. 

Introduction

Unbiased whole-genome genetic screening is a powerful approach for identifying genes involved in a given biological process and elucidating its mechanism. Therefore, it is widely used in various fields of biological research. Approximately 60% of Drosophila genes are conserved in humans^1,2, and ~75% of human disease genes have homologs in Drosophila³. Genetic screening is mainly divided into two types: loss of function (LOF) and gain of function (GOF). LOF genetic screens in Drosophila have played a critical role in elucidating mechanisms that govern nearly every aspect of biology. However, the majority of Drosophila genes do not have obvious LOF phenotypes⁴, and therefore, GOF screening is an important method for studying the function of those genes^4,5.

The binary GAL4/UAS system is commonly used for tissue-specific gene expression in Drosophila⁶. In this system, the tissue specifically expresses yeast transcription activator GAL4 that binds to the GAL4 responsive element (UAS) and thereby activates transcription of the downstream genetic components (e.g., cDNA and ORF)⁶. To perform genome-wide GOF screens in Drosophila, we need to construct a genome-wide UAS-cDNA/ORF plasmid library and, subsequently, a transgenic UAS-cDNA/ORF library in Drosophila.

Construction of a genome-wide UAS-cDNA/ORF plasmid library by conventional methods from publicly available cDNA/ORF clones is time-consuming and laborious, as every gene requires individualized designs, including primer design and synthesis, polymerase chain reaction (PCR), and gel purification, sequencing, restriction digestion, and so on^7,8. Therefore, the construction of such a plasmid library is a rate-limiting step in creating a genome-wide transgenic UAS-cDNA/ORF library in Drosophila. Recently, we successfully solved this problem by developing a novel method, CRISPR-based modular assembly (CRISPRmass)⁹. The core of CRISPRmass is to manipulate the shared vector sequences of a plasmid library through a combination of gene editing technology and seamless cloning technology.

Here, we present a protocol for CRISPRmass, which includes massively parallel two-step test tube reactions followed by bacterial transformation. CRISPRmass is a simple, fast, efficient, and cost-effective method that, in principle, can be used for high-throughput construction of various plasmid libraries.

CRISPRmass strategy
The procedure of CRISPRmass starts with parallel two-step test tube reactions prior to Escherichia coli (E. coli) transformation (Figure 1). Step 1 is the cleavage of the identical vector backbones of the cDNA/ORF plasmids by Cas9/sgRNA. An ideal cleavage site is adjacent to the 5′ end of cDNA/ORF. The cleavage products do not have to be purified. Step 2 is the insertion of a vector-specific UAS module into the Cas9/sgRNA linearized cDNA/ORF plasmids by Gibson assembly (hereafter referred to as single step reaction), resulting in UAS-cDNA/ORF plasmids. The 5′ and 3′ terminal sequences of a UAS module overlap with those of the linearized cDNA/ORF plasmids, enabling the single step reaction.

The single step reaction products are directly subjected to E. coli transformation. Theoretically, only the desired UAS-cDNA/ORF colonies can grow on Luria-Bertani (LB) plates that contain selection antibiotics corresponding to the antibiotic resistance gene of the UAS module. The UAS module is composed of a core UAS module, an antibiotic resistance gene distinct from that of cDNA or ORF plasmids, and the 5′ and 3′ terminal sequences. A core UAS module comprises 10 copies of UAS, an Hsp70 minimal promoter, an attB sequence for phiC31-mediated genomic integration, and a mini-white transformation marker for Drosophila⁷.

Protocol

1. Determination of optimal Cas9/sgRNA cleavage site upstream of cDNA/ORF Preparation of cDNA or ORF plasmids with identical vector backbone Purchase cDNA/ORF clones that are available in public resources, such as the Drosophila genome resource center (DGRC, https://dgrc.bio.indiana.edu/) Gold Collection10,11, the mouse mammalian gene collection (MGC, https://genecollections.nci.nih.gov/MGC/), and the human CCSB-b…

Representative Results

We applied CRISPRmass to generate a genome-wide UAS-cDNA/ORF plasmid library using 3402 cDNA/ORF clones bearing pOT2 vector backbone from the DGRC Gold Collection. We randomly analyzed only one colony for each UAS-cDNA/ORF construct, and subsequent restriction analysis with PstI indicated that 98.6% of UAS-cDNA/ORF constructs were created successfully9. The rationale for using PstI for restriction analysis of UAS-cDNA/ORF constructs bearing pOT2 vector backbone is as follows. There are two PstI si…

Discussion

The most critical steps of CRISPRmass are the design of sgRNAs and the evaluation of sgRNAs. Selection of highly efficient sgRNAs for Cas9 is key to the success of CRISPRmass. If very few or no colonies are observed on the majority of antibiotic-containing LB plates after the transformation of E. coli with single-step reaction assembly products, check plasmid digestion by agarose gel electrophoresis. If plasmids are not well digested, check Cas9 activity, sgRNA degradation and plasmid quality; if plasmids are well digest…

Materials

Aluminum Cooling Block	Aikbbio	ADMK-0296	To perform bacterial transformation
DEPC-Treated Water	Invitrogen	AM9906
Gel Extraction Kit	Omega	D2500	To purify DNA from agarose gel
Gel Imaging System	Tanon	2500B
HiScribe T7 Quick High Yield RNA Synthesis Kit	NEB	E2050
NEBuilder HiFi DNA Assembly Master Mix	NEB	E2621
Plasmid Mini Kit	Omega	D6943	To isolate plasmid DNA from bacterial cells
Q5 Hot Start High-Fidelity 2x Master Mix	NEB	M0494
S. pyogenes Cas9	GenScript	Z03386
Shaking Incubator	Shanghai Zhichu	ZQLY-180V
T series Multi-Block Thermal Cycler	LongGene	T20	To perform PCR
Trans10 Chemically Competent Cell	TranGen BioTech	CD101
Ultraviolet spectrophotometer	Shimadzu	BioSpec-nano	To measure concentration of DNA or RNA