Limits...
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.


Screening and quantification strategy.A: Screening strategy. Cells were transfected with the library (coloured circles), and selected in puromycin to enrich for transfected cells followed by growth for 1, 4 and 10 days (d). sgRNAs were quantified by PCR and high throughput sequencing (HTS). B: Amplification of sgRNAs from cells. sgRNA sequences were amplified by PCR using common flanking sequences to obtain an 84 bp product. A second round of PCR was performed to add adaptors. These included the sequences required for amplification prior to sequencing (P5, light red, P3, green) and sequencing primer binding sites (seq1, dark red, seq2, orange) and included two barcodes (BC1, light blue, BC2, dark blue). BC1 is of variable length to increase sequencing library diversity.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4508376&req=5

fig4: Screening and quantification strategy.A: Screening strategy. Cells were transfected with the library (coloured circles), and selected in puromycin to enrich for transfected cells followed by growth for 1, 4 and 10 days (d). sgRNAs were quantified by PCR and high throughput sequencing (HTS). B: Amplification of sgRNAs from cells. sgRNA sequences were amplified by PCR using common flanking sequences to obtain an 84 bp product. A second round of PCR was performed to add adaptors. These included the sequences required for amplification prior to sequencing (P5, light red, P3, green) and sequencing primer binding sites (seq1, dark red, seq2, orange) and included two barcodes (BC1, light blue, BC2, dark blue). BC1 is of variable length to increase sequencing library diversity.

Mentions: In order to optimise protocols for screening using this library, we transfected S2R+ cells, selected in puromycin to enrich for transfected cells, and took samples of cells at various timepoints after selection (1, 4 and 10 days) (Fig. 4A). sgRNAs targeting genes which are essential for cell survival or growth would be expected to be depleted over time, and those important for cell death processes should show the opposite effect, and be enriched at later time points. We quantified the sgRNA population in each of these samples by PCR across the sgRNA sequences and HTS (Fig. 4B). A two-step strategy was used to first amplify the sgRNA sequence, and then add the appropriate adaptors for sequencing. In order to multiplex several libraries in one sequencing run, we added two barcodes, a variable length adaptor at the 5′ end to increase library complexity, and a second at the 3′ end.


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

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

Screening and quantification strategy.A: Screening strategy. Cells were transfected with the library (coloured circles), and selected in puromycin to enrich for transfected cells followed by growth for 1, 4 and 10 days (d). sgRNAs were quantified by PCR and high throughput sequencing (HTS). B: Amplification of sgRNAs from cells. sgRNA sequences were amplified by PCR using common flanking sequences to obtain an 84 bp product. A second round of PCR was performed to add adaptors. These included the sequences required for amplification prior to sequencing (P5, light red, P3, green) and sequencing primer binding sites (seq1, dark red, seq2, orange) and included two barcodes (BC1, light blue, BC2, dark blue). BC1 is of variable length to increase sequencing library diversity.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig4: Screening and quantification strategy.A: Screening strategy. Cells were transfected with the library (coloured circles), and selected in puromycin to enrich for transfected cells followed by growth for 1, 4 and 10 days (d). sgRNAs were quantified by PCR and high throughput sequencing (HTS). B: Amplification of sgRNAs from cells. sgRNA sequences were amplified by PCR using common flanking sequences to obtain an 84 bp product. A second round of PCR was performed to add adaptors. These included the sequences required for amplification prior to sequencing (P5, light red, P3, green) and sequencing primer binding sites (seq1, dark red, seq2, orange) and included two barcodes (BC1, light blue, BC2, dark blue). BC1 is of variable length to increase sequencing library diversity.
Mentions: In order to optimise protocols for screening using this library, we transfected S2R+ cells, selected in puromycin to enrich for transfected cells, and took samples of cells at various timepoints after selection (1, 4 and 10 days) (Fig. 4A). sgRNAs targeting genes which are essential for cell survival or growth would be expected to be depleted over time, and those important for cell death processes should show the opposite effect, and be enriched at later time points. We quantified the sgRNA population in each of these samples by PCR across the sgRNA sequences and HTS (Fig. 4B). A two-step strategy was used to first amplify the sgRNA sequence, and then add the appropriate adaptors for sequencing. In order to multiplex several libraries in one sequencing run, we added two barcodes, a variable length adaptor at the 5′ end to increase library complexity, and a second at the 3′ end.

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.