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A New Age in Functional Genomics Using CRISPR/Cas9 in Arrayed Library Screening


A New Age in Functional Genomics Using CRISPR/Cas9 in Arrayed Library Screening

Alexander Agrotis et al. Front Genet.


CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

Keywords: CRISPR; Cas9/sgRNA; high-content imaging; high-throughput screening; knockdown; siRNA.


The mechanism of genome editing using CRISPR/Cas9. The genomic DNA target, which must lie adjacent to a protospacer adjacent motif (PAM), is specified by a 20 nt user-generated guide sequence in the sgRNA or crRNA. The Streptococcus pyogenes PAM is shown. In the cell nucleus, Cas9 protein associates with the sgRNA or crRNA/tracrRNA and binds to the target sequence, cleaving both strands of the DNA at the site 3 nt upstream of the PAM. Cleavage results in a DSB which is repaired by host-mediated DNA repair mechanisms. In the absence of a repair template, error-prone NHEJ occurs which may lead to the formation of random short indels and thus frameshift mutations and disruption of gene function, and this represents the main method of CRISPR-mediated gene knockout generation. If an artificial repair template is provided, for example on a plasmid containing a sequence of interest flanked by homology arms, then HDR may occur, leading to the introduction of an exogenous DNA sequence at a specified genomic location. This is the basis for performing gene knock-in, tagging, and precise pre-specified insertions or deletions using CRISPR. If catalytically inactive Cas9 is used instead of wild-type Cas9, then the protein simply binds to the target locus and does not cleave the DNA.
General workflow for screening using CRISPR/Cas9 in pooled versus arrayed approaches. In pooled screening, the viral sgRNA library is delivered to a single vessel of cells at low multiplicity of infection (MOI), before selections for transduced cells and specific phenotypes are carried out. The output of the screen is derived from deep sequencing of genomic DNA from the selected versus control cell populations, providing a measure of enrichment or depletion of each sgRNA in response to selection. In arrayed library screening, more library types and delivery methods are available since sgRNAs are delivered to discreet populations of cells grown in an arrayed format, preventing an individual cell from possessing multiple sgRNAs with different targets. There may be selection steps and treatments involved, but this can vary depending on the screen. Phenotypes are identified rather than necessarily being selected for, since the sgRNA responsible for each phenotype is known based on well location in the original annotated library. The final output for this method is a ranked phenotypic measure for each sgRNA delivered in the screen, and it may be chosen to detect multiple phenotypes in a single screen. In both methods, Cas9 can be stably expressed in the cells or co-delivered with the library. Key protocol steps that refer to both screening methods are indicated in pale yellow boxes.

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