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A genome-wide CRISPR library for high-throughput genetic screening in Drosophila cells.

Bassett AR, Kong L, Liu JL - J Genet Genomics (2015)

Bottom Line: The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes.Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS).

View Article: PubMed Central - PubMed

Affiliation: MRC Functional Genomics Unit, University of Oxford, Department of Physiology, Anatomy and Genetics, South Parks Road, Oxford, OX1 3PT, United Kingdom; Genome Engineering Oxford, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom. Electronic address: andrew.bassett@path.ox.ac.uk.

No MeSH data available.


Analysis of cloned sgRNA library.A: Pie chart of the sgRNA sequences represented in the cloned library. Those sequences represented by 0 read (purple), 1 read (dark blue), 2–5 reads (mid blue) and more than 5 reads (light blue) are indicated. B: Histogram of the number of sgRNAs per gene in the cloned library. Total number of genes with designs is 13,668, and the number of genes with at least one sgRNA is 13,501. C: Example of cloned sgRNA. Screenshot from UCSC browser shows designed sgRNAs and cloned sgRNAs at a typical gene (CG2219).
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fig3: Analysis of cloned sgRNA library.A: Pie chart of the sgRNA sequences represented in the cloned library. Those sequences represented by 0 read (purple), 1 read (dark blue), 2–5 reads (mid blue) and more than 5 reads (light blue) are indicated. B: Histogram of the number of sgRNAs per gene in the cloned library. Total number of genes with designs is 13,668, and the number of genes with at least one sgRNA is 13,501. C: Example of cloned sgRNA. Screenshot from UCSC browser shows designed sgRNAs and cloned sgRNAs at a typical gene (CG2219).

Mentions: We then assessed the coverage of sgRNAs in the library by high throughput sequencing (HTS) of a PCR product across the sgRNA sequences, at a depth of 827,527 mapped reads. This showed that 40,279 sgRNAs were represented by at least one sequencing read (Fig. 3A), and 21,805 sgRNAs by more than 5 reads. Analysis of the genes represented in the library showed that 13,501 genes (98.8%) were represented by at least one sgRNA and 8989 genes (65.8%) were targeted by 3 or more sgRNAs (Fig. 3B). This is of particular note since recent data suggest that the specificity of sgRNA screens can be improved by selecting those genes where similar effects are seen for multiple independent sgRNAs (Shalem et al., 2014; Koike-Yusa et al., 2014). A typical distribution of sgRNAs across a gene is shown in Fig. 3C, and BED files of both the total 68,340 sgRNA set and cloned 40,279 sgRNA set are available (Supplementary Data), which can be uploaded directly to a genome browser.


A genome-wide CRISPR library for high-throughput genetic screening in Drosophila cells.

Bassett AR, Kong L, Liu JL - J Genet Genomics (2015)

Analysis of cloned sgRNA library.A: Pie chart of the sgRNA sequences represented in the cloned library. Those sequences represented by 0 read (purple), 1 read (dark blue), 2–5 reads (mid blue) and more than 5 reads (light blue) are indicated. B: Histogram of the number of sgRNAs per gene in the cloned library. Total number of genes with designs is 13,668, and the number of genes with at least one sgRNA is 13,501. C: Example of cloned sgRNA. Screenshot from UCSC browser shows designed sgRNAs and cloned sgRNAs at a typical gene (CG2219).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508376&req=5

fig3: Analysis of cloned sgRNA library.A: Pie chart of the sgRNA sequences represented in the cloned library. Those sequences represented by 0 read (purple), 1 read (dark blue), 2–5 reads (mid blue) and more than 5 reads (light blue) are indicated. B: Histogram of the number of sgRNAs per gene in the cloned library. Total number of genes with designs is 13,668, and the number of genes with at least one sgRNA is 13,501. C: Example of cloned sgRNA. Screenshot from UCSC browser shows designed sgRNAs and cloned sgRNAs at a typical gene (CG2219).
Mentions: We then assessed the coverage of sgRNAs in the library by high throughput sequencing (HTS) of a PCR product across the sgRNA sequences, at a depth of 827,527 mapped reads. This showed that 40,279 sgRNAs were represented by at least one sequencing read (Fig. 3A), and 21,805 sgRNAs by more than 5 reads. Analysis of the genes represented in the library showed that 13,501 genes (98.8%) were represented by at least one sgRNA and 8989 genes (65.8%) were targeted by 3 or more sgRNAs (Fig. 3B). This is of particular note since recent data suggest that the specificity of sgRNA screens can be improved by selecting those genes where similar effects are seen for multiple independent sgRNAs (Shalem et al., 2014; Koike-Yusa et al., 2014). A typical distribution of sgRNAs across a gene is shown in Fig. 3C, and BED files of both the total 68,340 sgRNA set and cloned 40,279 sgRNA set are available (Supplementary Data), which can be uploaded directly to a genome browser.

Bottom Line: The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes.Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS).

View Article: PubMed Central - PubMed

Affiliation: MRC Functional Genomics Unit, University of Oxford, Department of Physiology, Anatomy and Genetics, South Parks Road, Oxford, OX1 3PT, United Kingdom; Genome Engineering Oxford, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom. Electronic address: andrew.bassett@path.ox.ac.uk.

No MeSH data available.