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A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

Agrotis A, Ketteler R - Front Genet (2015)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London UK.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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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Figure 2: 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.

Mentions: A growing number of published studies have utilized CRISPR technology for screening (see Table 3 for a comparison). CRISPR screening can be performed using pooled library approaches coupled to positive or negative selection, or alternatively arrayed libraries (Figure 2). To date, all studies have employed a pooled library approach, and whilst performed primarily as a proof-of-principle, these have uncovered novel genes that enhance or suppress simple phenotypes such as toxicity-induced cell death (Gilbert et al., 2014; Koike-Yusa et al., 2014; Shalem et al., 2014; Wang et al., 2014; Zhou et al., 2014; Konermann et al., 2015). They have also helped confirm important parameters of effective sgRNA design, which will undoubtedly assist the production of future libraries. A summary of these design considerations is listed in a recommended technical resource (Malina et al., 2014).


A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

Agrotis A, Ketteler R - Front Genet (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585242&req=5

Figure 2: 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.
Mentions: A growing number of published studies have utilized CRISPR technology for screening (see Table 3 for a comparison). CRISPR screening can be performed using pooled library approaches coupled to positive or negative selection, or alternatively arrayed libraries (Figure 2). To date, all studies have employed a pooled library approach, and whilst performed primarily as a proof-of-principle, these have uncovered novel genes that enhance or suppress simple phenotypes such as toxicity-induced cell death (Gilbert et al., 2014; Koike-Yusa et al., 2014; Shalem et al., 2014; Wang et al., 2014; Zhou et al., 2014; Konermann et al., 2015). They have also helped confirm important parameters of effective sgRNA design, which will undoubtedly assist the production of future libraries. A summary of these design considerations is listed in a recommended technical resource (Malina et al., 2014).

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London UK.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus