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.


Related in: MedlinePlus

Design of a genome-wide sgRNA library.A: Strategy for library design. Fragments of coding exons shared between the maximum number of transcriptional isoforms were selected, and all possible sgRNAs of the format N20NGG were designed on both strands. Exons (blue boxes), transcriptional start sites (arrows) and untranslated regions (thick blue lines) are indicated. sgRNAs were selected based on the absence of any off-targets with less than three mismatches, and their position early in the coding sequence. Five non-overlapping sequences were selected. B: Example of designs. A screenshot from the UCSC browser shows designs for a typical gene.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig1: Design of a genome-wide sgRNA library.A: Strategy for library design. Fragments of coding exons shared between the maximum number of transcriptional isoforms were selected, and all possible sgRNAs of the format N20NGG were designed on both strands. Exons (blue boxes), transcriptional start sites (arrows) and untranslated regions (thick blue lines) are indicated. sgRNAs were selected based on the absence of any off-targets with less than three mismatches, and their position early in the coding sequence. Five non-overlapping sequences were selected. B: Example of designs. A screenshot from the UCSC browser shows designs for a typical gene.

Mentions: The Cas9 protein induces a double strand break (DSB) at its complimentary target site in the genome that is repaired by the endogenous repair pathways in the cell (Rouet et al., 1994). In most cell types, non-homologous end joining (NHEJ) repair predominates over homologous recombination (HR) (Shrivastav et al., 2008). Since NHEJ is somewhat error-prone, this results in small insertion and deletion mutations at the cleavage site, which can be employed to cause frameshifts in protein-coding sequence (Bibikova et al., 2002). We therefore obtained the sequences of all protein coding exons from Flybase (release 5.57), and extracted exonic regions from those that are shared between the maximum number of transcripts in order to maximise the effect of the gene knockout (Fig. 1A).


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

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

Design of a genome-wide sgRNA library.A: Strategy for library design. Fragments of coding exons shared between the maximum number of transcriptional isoforms were selected, and all possible sgRNAs of the format N20NGG were designed on both strands. Exons (blue boxes), transcriptional start sites (arrows) and untranslated regions (thick blue lines) are indicated. sgRNAs were selected based on the absence of any off-targets with less than three mismatches, and their position early in the coding sequence. Five non-overlapping sequences were selected. B: Example of designs. A screenshot from the UCSC browser shows designs for a typical gene.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig1: Design of a genome-wide sgRNA library.A: Strategy for library design. Fragments of coding exons shared between the maximum number of transcriptional isoforms were selected, and all possible sgRNAs of the format N20NGG were designed on both strands. Exons (blue boxes), transcriptional start sites (arrows) and untranslated regions (thick blue lines) are indicated. sgRNAs were selected based on the absence of any off-targets with less than three mismatches, and their position early in the coding sequence. Five non-overlapping sequences were selected. B: Example of designs. A screenshot from the UCSC browser shows designs for a typical gene.
Mentions: The Cas9 protein induces a double strand break (DSB) at its complimentary target site in the genome that is repaired by the endogenous repair pathways in the cell (Rouet et al., 1994). In most cell types, non-homologous end joining (NHEJ) repair predominates over homologous recombination (HR) (Shrivastav et al., 2008). Since NHEJ is somewhat error-prone, this results in small insertion and deletion mutations at the cleavage site, which can be employed to cause frameshifts in protein-coding sequence (Bibikova et al., 2002). We therefore obtained the sequences of all protein coding exons from Flybase (release 5.57), and extracted exonic regions from those that are shared between the maximum number of transcripts in order to maximise the effect of the gene knockout (Fig. 1A).

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.


Related in: MedlinePlus