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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.


Cloning of sgRNA library.A: sgRNA expression vector. sgRNAs (blue) are expressed from a Drosophila U6:2 promoter, along with the Cas9 protein from an Actin-5C promoter. Cas9 (red box) contains N- and C-terminal nuclear localisation sequences (NLS, grey oval), and is expressed as a bicistronic transcript with a puromycin N-acetyltransferase gene (purple oval) separated by a viral 2A peptide (black oval). An SV40 transcriptional terminator is also included (SV40 term). B: Oligo synthesis. sgRNA sequences were synthesised with common 5′ and 3′adaptors, and amplified by PCR followed by digestion with restriction enzymes and cloned into the expression vector. C: Cloning strategy for sgRNAs. The synthesised oligonucleotides were amplified by PCR using common adaptor sequences, and digested with the BspQ I restriction enzyme, followed by ligation into a similarly digested expression vector. The first base transcribed by the dU6:2 promoter (G) is indicated by an arrow.
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fig2: Cloning of sgRNA library.A: sgRNA expression vector. sgRNAs (blue) are expressed from a Drosophila U6:2 promoter, along with the Cas9 protein from an Actin-5C promoter. Cas9 (red box) contains N- and C-terminal nuclear localisation sequences (NLS, grey oval), and is expressed as a bicistronic transcript with a puromycin N-acetyltransferase gene (purple oval) separated by a viral 2A peptide (black oval). An SV40 transcriptional terminator is also included (SV40 term). B: Oligo synthesis. sgRNA sequences were synthesised with common 5′ and 3′adaptors, and amplified by PCR followed by digestion with restriction enzymes and cloned into the expression vector. C: Cloning strategy for sgRNAs. The synthesised oligonucleotides were amplified by PCR using common adaptor sequences, and digested with the BspQ I restriction enzyme, followed by ligation into a similarly digested expression vector. The first base transcribed by the dU6:2 promoter (G) is indicated by an arrow.

Mentions: In total we designed 68,340 sgRNAs, covering 13,668 genes (approximately 78% of all Drosophila genes) and a typical distribution of these guides is indicated in Fig. 1B. These sgRNAs had common adaptors added, and were synthesised as a large pool, followed by PCR amplification using the common sequences, and digestion with a restriction enzyme to release the sgRNA sequences. These sequences were purified and cloned into an S2 expression vector (Bassett et al., 2014), which expresses the sgRNA from a Drosophila U6:2 promoter, and the Cas9 protein with N- and C-terminal nuclear localisation signals under the control of the actin 5C promoter (Fig. 2A and B). Additionally, the vector contains a puromycin N-acetyltransferase gene to allow selection in S2 cells. Cloning of the sgRNAs uses the type IIS restriction enzyme BspQ I that allows scarless integration of the 20 nt target sequence (Fig. 2C). In order to maintain representation of the library, a total of approximately 7 million bacterial colonies were generated, representing at least a 100-fold coverage of the library.


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

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

Cloning of sgRNA library.A: sgRNA expression vector. sgRNAs (blue) are expressed from a Drosophila U6:2 promoter, along with the Cas9 protein from an Actin-5C promoter. Cas9 (red box) contains N- and C-terminal nuclear localisation sequences (NLS, grey oval), and is expressed as a bicistronic transcript with a puromycin N-acetyltransferase gene (purple oval) separated by a viral 2A peptide (black oval). An SV40 transcriptional terminator is also included (SV40 term). B: Oligo synthesis. sgRNA sequences were synthesised with common 5′ and 3′adaptors, and amplified by PCR followed by digestion with restriction enzymes and cloned into the expression vector. C: Cloning strategy for sgRNAs. The synthesised oligonucleotides were amplified by PCR using common adaptor sequences, and digested with the BspQ I restriction enzyme, followed by ligation into a similarly digested expression vector. The first base transcribed by the dU6:2 promoter (G) is indicated by an arrow.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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fig2: Cloning of sgRNA library.A: sgRNA expression vector. sgRNAs (blue) are expressed from a Drosophila U6:2 promoter, along with the Cas9 protein from an Actin-5C promoter. Cas9 (red box) contains N- and C-terminal nuclear localisation sequences (NLS, grey oval), and is expressed as a bicistronic transcript with a puromycin N-acetyltransferase gene (purple oval) separated by a viral 2A peptide (black oval). An SV40 transcriptional terminator is also included (SV40 term). B: Oligo synthesis. sgRNA sequences were synthesised with common 5′ and 3′adaptors, and amplified by PCR followed by digestion with restriction enzymes and cloned into the expression vector. C: Cloning strategy for sgRNAs. The synthesised oligonucleotides were amplified by PCR using common adaptor sequences, and digested with the BspQ I restriction enzyme, followed by ligation into a similarly digested expression vector. The first base transcribed by the dU6:2 promoter (G) is indicated by an arrow.
Mentions: In total we designed 68,340 sgRNAs, covering 13,668 genes (approximately 78% of all Drosophila genes) and a typical distribution of these guides is indicated in Fig. 1B. These sgRNAs had common adaptors added, and were synthesised as a large pool, followed by PCR amplification using the common sequences, and digestion with a restriction enzyme to release the sgRNA sequences. These sequences were purified and cloned into an S2 expression vector (Bassett et al., 2014), which expresses the sgRNA from a Drosophila U6:2 promoter, and the Cas9 protein with N- and C-terminal nuclear localisation signals under the control of the actin 5C promoter (Fig. 2A and B). Additionally, the vector contains a puromycin N-acetyltransferase gene to allow selection in S2 cells. Cloning of the sgRNAs uses the type IIS restriction enzyme BspQ I that allows scarless integration of the 20 nt target sequence (Fig. 2C). In order to maintain representation of the library, a total of approximately 7 million bacterial colonies were generated, representing at least a 100-fold coverage of the library.

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.