An sgRNA library is a collection of genes containing millions of sgRNA sequences. It can be utilized to knock out human whole genes, pathway genes and specific gene families in high throughput, and can rapidly screen out genes related to certain disease phenotypes and traits, which plays an important role in gene function research, disease diagnosis, molecular therapy and drug development. Since sgRNA libraries can be used for genome-wide screening, regardless of sequence, cell or species, and can quickly and accurately find genes related to the target phenotypes, they are favored by many researchers because of their high editing accuracy and target hit rate.

sgRNA libraries can be categorized into CRISPRko knockdown library, CRISPRa activation library, and CRISPRi interference library according to their effects.

Fig. 1 The CRISPR, CRISPRi, and CRISPRa systems.

CRISPRko Knockout Library

The target sequence was specifically recognized by sgRNA, and the Cas9 endonuclease was directed to cleave the outer 3 nucleotides upstream of the PAM of the target sequence, resulting in a double-stranded break in the DNA at the target site, and the knockdown was achieved by introducing a shifted mutation through the non-homologous end-joining (NHEJ) DNA repair mechanism.

By using one sgRNA, CRISPR-ko of a single target can be achieved. However, by using multiple sgRNAs designed to target distinct genes, the investigator can generate a sgRNA library, which allows high-throughput genetic screens. Construction of a library containing sgRNAs against all protein-coding genes enables a genome-wide screen, whereas a smaller library containing sgRNAs against preselected genes enables targeted assessment of a specific gene set. During a CRISPR screen, the sgRNA library is introduced into a cell population in a manner such that each cell receives only one sgRNA. As a result, each cell within the bulk population undergoes a single knockout event, but the targeted elements differ between cells.

To improve knockout efficiency, Creative Bioigene offers a number of ready-to-use gene editing kits to help customers perform efficient gene editing.

View more of our gene editing kit products.

CRISPRa Activation Library

Transcription activation functional domain fusion with catalytically inactive Cas9 (dCas9) to activate target sites (usually promoter regions and other non-coding regulatory regions) to regulate gene expression, mainly used for gain-of-function screening of coding genes or regulatory function screening of non-coding regions.

CRISPRi Interference Library

Transcriptional deterrent fusion dCas9 to inhibit target sites (usually promoter regions and other non-coding regulatory regions) to regulate gene expression, loss-of-function screens of coding genes or regulatory function screens of non-coding regions.

To help researchers succeed in gene editing, Creative Biogene offers custom CRISPR sgRNA vectors. Our customized vectors can be used in a wide range of cells, including both easy and hard-to-transfect cells, covering the entire genomes of humans, mice and rats.

sgRNA Library Construction Procedure

The sgRNA library construction mainly involves designing and synthesizing multiple sgRNAs by targeting the same gene, establishing knockout libraries with the CRISPR system, and screening the relevant genes by functional screening and sequencing analysis.

• Design and synthesize sgRNA

Design of gRNAs, 3-6 per gene. Synthesize sgRNA by microarray synthesis.

• Library construction

After PCR amplification, construct the plasmid, transform the extracted plasmid, analyze the abundance and balance by NGS sequencing, and successfully construct the sgRNA library.

• Lentiviral packaging

The constructed plasmid was lentivirally packaged, and the titer was detected to obtain the library virus.

• Cell Infection and screening

After the library lentivirus infects cells, a resistance (antidote) screen is performed to identify candidate genes.

• Target gene identification

Target gene identification by monoclonal cell culture and amplicon sequencing.

Advantages of sgRNA Library

Due to the cumbersome process of sgRNA library construction and many influencing factors, it is easy to lead to long time-consuming repeat experiments and unsatisfactory results. In order to speed up the construction process and ensure the correct editing rate and target hit rate, Creative Biogene provides a one-stop solution for the construction and screening of sgRNA libraries for the whole human genome, specific signaling pathways or customer-specified genes, which includes services such as sgRNA library design, sgRNA synthesis, lentiviral packaging, cell line construction, CRISPR Screen (sgRNA) sequencing analysis, etc. for sgRNA library construction. library) sequencing analysis, and other services to ensure the construction of sgRNA libraries. Our sgRNA libraries have the following advantages:

• Wide range of applications, independent of gene sequences, cells and species.
• Quickly and accurately find genes and gene clusters associated with a certain phenotype.
• Be used for genome-wide screening, which is conducive to the discovery of new targets.
• Low false-positive rate of editing effect and high target hit rate.

Application of sgRNA Library in Tumor Direction

Identification of tumor driver genes and targeted therapy against oncogenes

The regulation of cell growth by genes is like driving a car, with two main types of genes regulating it: "refueling genes" and "brake genes". The "refueling genes" are responsible for maintaining the growth rate, so that the cells are constantly renewed; the "brake genes" are responsible for slowing down or terminating the growth of the cells, so that the new cells can replace the original function. In patients with malignant tumors, mutations in certain "fueling genes" or "brake genes" instead play a driving role and have a significant impact on cancer development and progression, and these genes are known as tumor driver genes (Driver genes). The most common ones are TP53, PIK3CA, KMT2C, ARID1A, KMT2D, LRP1B, PTEN, RB1, FAT4, KRAS and so on.

Fig. 2 Schematic diagram of the CRISPR-Cas9 library positive and negative selection workflow.

CRlSPR/Cas9 gRNA libraries are an excellent tool for high-throughput gene inactivation because they can simultaneously target different genes across the genome. The article "CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives" published in the journal Theranostics describes the recent emergence of high-throughput CRISPR-Cas9 gRNA library genomic screening technology and its principle of operation, and highlights the advantages of its various applications in anti-cancer drug discovery, including functional and target identification, non-coding RNA information, the role of small molecules, and drug target discovery.

Screening of synthetic lethal targets for precision therapy

Synthetic lethality (SL), the simultaneous inactivation of two non-lethal genes leading to cell death, has triggered the industry's attention as an emerging area of oncology therapeutic development with the advancement of new drugs based on this mechanism, which is usually categorized into two main groups: unconditional/primitive SL and conditional SL.

How to use CRISPR/Cas9 sgRNA libraries to find more gene pairs with synthetic lethal effects? There are two general logics at present:

(1) Direct design of double-targeting gRNA vectors, i.e., targeting two genes simultaneously in a single cell to screen for gene pairs with synthetic lethal effects;

(2) Library screening on a single oncogene knockout cell line to screen for synthetic lethality with the knockout oncogene.

Searching for tumor drug resistance related genes

It is well known that tumor drug resistance is one of the major causes of tumor treatment failure, which greatly limits the selection and use of cancer drugs, so exploring the mechanism of drug resistance in tumors is of great clinical significance (Figure 7). Drug resistance can be classified as endogenous (or ab initio) resistance (the presence of drug resistance factors in tumor cells before drug therapy) or acquired resistance (resistance develops during drug therapy), and, an important feature of drug resistance is that resistance to one drug may lead to resistance to other drugs, which is multidrug resistance (MDR).

The study confirmed that after positive screening of CRISPR/Cas9 sgRNA libraries, the majority of cells die due to drug sensitivity, and some cells survive due to drug resistance resulting from specific gene knockdowns. By detecting the gRNAs carried by the surviving cells, the genes associated with the development of drug resistance can be screened out, bringing hope for overcoming drug resistance in tumors as well as for effective cancer treatment.