Everything You Need To Know About CRISPR gRNA Library Screening
CRISPR screen library is an important tool to identify which genes are important for the target phenotype. The underlying genetic factors of many diseases are unclear. Studying the role of a single gene or entire pathway in specific disease processes requires a lot of experiments. Although CRISPR is not the first method to conduct so-called "forward genetic screening", it is certainly the most powerful one. In this article, we will introduce how to use the CRISPR gRNA library and CRISPR Cas9 cell line for genome wide screening.
▍ What makes CRISPR so advantageous?
One of the main advantages of CRISPR/Cas9 compared to previous genome editing technologies is its simplicity and versatility. CRISPR/Cas9 consists of two parts: a nonspecific endonuclease (Cas9) and a single stranded guide RNA (gRNA). The~20 nucleotide target sequence in gRNA is defined by researchers and can easily guide Cas9 to almost any genomic loci, provided that the target is unique compared to the rest of the genome sequence and is located at the 5 'end of the protospacer adjacent motif (PAM). The co-delivery of wild-type Cas9 and gRNA can cause double strand breaks on the target DNA, and when repaired through non-homologous end-joining (NHEJ), mutations can result in the loss of function of the target gene. CRISPR can also activate or knockdown target genes without modifying the genome, ie CRISPRa and CRISPRi.
▍ CRISPR screen library for whole genome screening
The goal of genome wide screening experiments is to generate and screen mutant cell populations to identify genes associated with specific phenotypes. Because CRISPR has a wide range of potential target sequences and convenient construction of gRNA plasmids, it is easy to use for genome wide screening. CRISPR genome wide screening experiments typically use lentiviruses to deliver multiple gRNAs to Cas9 expressing stable cells. A lentivirus CRISPR library (abbreviated as "CRISPR library") with a collection of multiple gRNAs consists of a heterogeneous population of gRNAs containing lentivirus transfer vectors, each vector targeting a specific gene. CRISPR libraries knockout, activate, or interfere target genes by combining gRNA libraries with Cas9 or dCas9.
▍ How to choose a CRISPR library
When choosing a CRISPR library, several aspects need to be considered:
1. What species are you working on? Currently, commercialized CRISPR libraries can target genes of mouse, human, fly, E.coli, and T.gondii.
2. What genetic modification do you want to perform? Commercialized libraries can be used for gene knockout, activation, repression, and barcoding.
3. Is the goal only for each gene in the genome, or for a specific class of genes? The commercialized library includes a whole genome CRISPR library and a series of defined libraries targeting specific gene categories.
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▍ Steps of CRISPR library screening
Using a CRISPR library for forward genetic screening is a multi-step process.
Cells are then infected with a lentivirus CRISPR library to produce a heterogeneous mutant cell population, with each cell or group of cells containing different gene mutations. The MOI is usually <1.The library can be whether gRNA and Cas9 all in one (one plasmid system) or gRNA and Cas9 separate plasmids (two plasmids system).It is highly recommend to use two plasmids system, becasue the size of gRNA and Cas9 together is too large for lentivirus packaging, which means it is difficult to get a high titer lentivirus library for one plasmid system. With two plasmids system, simply infect the gRNAs lentivirus to a Cas9 stable cell line can effectively screen for genes.
Use drug screening or fluorescence based sorting to enrich mutant cells and screen for specific phenotypes. For example, mutant cells can be screened for drugs to identify genes that cause resistance. "Mutant cells were treated with drugs of interest, and the distribution of gRNA in drug resistant populations was analyzed compared to untreated control groups.". In this case, the "enriched" gRNA corresponds to the gene that produces resistance at the time of the mutation. The findings of such experiments can reveal the mechanism by which cells acquire drug resistance and identify future therapeutic targets for diseases that further indicate abnormal cell growth, such as cancer.
▍ Tips for successful CRISPR screen
▍ Avaliable CRISPR Cas9 cell lines for gRNA library screening
▍ Reference