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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

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.
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Figure 1: 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.

Mentions: The type II CRISPR system from Streptococcus pyogenes has been adapted for targeted genome editing in eukaryotic cells (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013a,b). In a three-component system that is reminiscent of the natural system, the two RNA components (crRNA and tracrRNA) are introduced into cells or ectopically expressed along with a codon-optimized Cas9. When the crRNA and tracrRNA are artificially fused as a fully functional single “guide” (sg) RNA this becomes an even simpler two-component system, consisting of just the sgRNA and Cas9 (Jinek et al., 2013). The principle of targeting in both systems remains the same. The host genome of interest is targeted for disruption by identifying a suitable PAM motif and designing a targeting sequence specific for the adjacent region, to incorporate into the crRNA or sgRNA. This allows Cas9 to be recruited to the desired locus to exert its function, most commonly by forming double-strand breaks (DSB) in the DNA in the case of “wild-type” Cas9 (Figure 1). The presence of a DSB initiates host-mediated cellular repair pathways; in the absence of a repair template, non-homologous end-joining (NHEJ) is carried out. This error-prone mechanism frequently causes insertions or deletions (indels) in the DNA that can result in frame-shifting and disruption of the gene (Jinek et al., 2013). If a repair template is available then homology-directed repair (HDR) may occur. This principle is exploited for performing knock-ins using CRISPR; by providing an artificial repair template in addition to the two or three CRISPR components, one can efficiently introduce recombinant DNA into the genome at a defined location.


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

Agrotis A, Ketteler R - Front Genet (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: 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.
Mentions: The type II CRISPR system from Streptococcus pyogenes has been adapted for targeted genome editing in eukaryotic cells (Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013a,b). In a three-component system that is reminiscent of the natural system, the two RNA components (crRNA and tracrRNA) are introduced into cells or ectopically expressed along with a codon-optimized Cas9. When the crRNA and tracrRNA are artificially fused as a fully functional single “guide” (sg) RNA this becomes an even simpler two-component system, consisting of just the sgRNA and Cas9 (Jinek et al., 2013). The principle of targeting in both systems remains the same. The host genome of interest is targeted for disruption by identifying a suitable PAM motif and designing a targeting sequence specific for the adjacent region, to incorporate into the crRNA or sgRNA. This allows Cas9 to be recruited to the desired locus to exert its function, most commonly by forming double-strand breaks (DSB) in the DNA in the case of “wild-type” Cas9 (Figure 1). The presence of a DSB initiates host-mediated cellular repair pathways; in the absence of a repair template, non-homologous end-joining (NHEJ) is carried out. This error-prone mechanism frequently causes insertions or deletions (indels) in the DNA that can result in frame-shifting and disruption of the gene (Jinek et al., 2013). If a repair template is available then homology-directed repair (HDR) may occur. This principle is exploited for performing knock-ins using CRISPR; by providing an artificial repair template in addition to the two or three CRISPR components, one can efficiently introduce recombinant DNA into the genome at a defined location.

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