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High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.

Ramlee MK, Yan T, Cheung AM, Chuah CT, Li S - Sci Rep (2015)

Bottom Line: Recent advances in the engineering of sequence-specific synthetic nucleases provide enormous opportunities for genetic manipulation of gene expression in order to study their cellular function in vivo.Here, we describe an efficient method of genotyping clonal CRISPR/Cas9-mediated mutants in a high-throughput manner involving the use of a direct lysis buffer to extract crude genomic DNA straight from cells in culture, and fluorescent PCR coupled with capillary gel electrophoresis.This technique also allows for genotyping of multiplexed gene targeting in a single clone.

View Article: PubMed Central - PubMed

Affiliation: Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore 169857.

ABSTRACT
Recent advances in the engineering of sequence-specific synthetic nucleases provide enormous opportunities for genetic manipulation of gene expression in order to study their cellular function in vivo. However, current genotyping methods to detect these programmable nuclease-induced insertion/deletion (indel) mutations in targeted human cells are not compatible for high-throughput screening of knockout clones due to inherent limitations and high cost. Here, we describe an efficient method of genotyping clonal CRISPR/Cas9-mediated mutants in a high-throughput manner involving the use of a direct lysis buffer to extract crude genomic DNA straight from cells in culture, and fluorescent PCR coupled with capillary gel electrophoresis. This technique also allows for genotyping of multiplexed gene targeting in a single clone. Overall, this time- and cost-saving technique is able to circumvent the limitations of current genotyping methods and support high-throughput screening of nuclease-induced mutants.

No MeSH data available.


Related in: MedlinePlus

Schematic of high-throughput genotyping technique via fluorescent PCR-capillary gel electrophoresis.First, cells are transfected with plasmids expressing Cas9-GFP fusion protein and individual sgRNA. Two days later, GFP-positive cells are sorted and plated onto 10-cm dishes. When individual colonies of cells are visible, they are picked and arrayed on 96-well plates. When the arrayed cells reach ~80% confluence, they are lysed directly using Direct-Lyse buffer and the crude lysate is used to amplify the genomic region containing the expected indel site using fluorophore-labelled primers. The labelled amplicons are resolved via capillary gel electrophoresis and successful mutants are identified by shifts in fragment size with respect to wildtype fragment. Putative knock-out clones are expanded and validated via Sanger sequencing, quantitative RT-PCR and/or Western blot analysis.
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f1: Schematic of high-throughput genotyping technique via fluorescent PCR-capillary gel electrophoresis.First, cells are transfected with plasmids expressing Cas9-GFP fusion protein and individual sgRNA. Two days later, GFP-positive cells are sorted and plated onto 10-cm dishes. When individual colonies of cells are visible, they are picked and arrayed on 96-well plates. When the arrayed cells reach ~80% confluence, they are lysed directly using Direct-Lyse buffer and the crude lysate is used to amplify the genomic region containing the expected indel site using fluorophore-labelled primers. The labelled amplicons are resolved via capillary gel electrophoresis and successful mutants are identified by shifts in fragment size with respect to wildtype fragment. Putative knock-out clones are expanded and validated via Sanger sequencing, quantitative RT-PCR and/or Western blot analysis.

Mentions: Current methods of genotyping engineered nuclease-mediated mutations are not optimal for high-throughput screening of individual knockout clones due to inherent limitations and/or high cost. We thus sought to determine whether fluorescent PCR coupled with capillary gel electrophoresis is able to accurately genotype mutant clones in a high-throughput and cost-effective manner and hence circumvent the abovementioned limitations (see Fig. 1 for schematic of protocol).


High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.

Ramlee MK, Yan T, Cheung AM, Chuah CT, Li S - Sci Rep (2015)

Schematic of high-throughput genotyping technique via fluorescent PCR-capillary gel electrophoresis.First, cells are transfected with plasmids expressing Cas9-GFP fusion protein and individual sgRNA. Two days later, GFP-positive cells are sorted and plated onto 10-cm dishes. When individual colonies of cells are visible, they are picked and arrayed on 96-well plates. When the arrayed cells reach ~80% confluence, they are lysed directly using Direct-Lyse buffer and the crude lysate is used to amplify the genomic region containing the expected indel site using fluorophore-labelled primers. The labelled amplicons are resolved via capillary gel electrophoresis and successful mutants are identified by shifts in fragment size with respect to wildtype fragment. Putative knock-out clones are expanded and validated via Sanger sequencing, quantitative RT-PCR and/or Western blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4620477&req=5

f1: Schematic of high-throughput genotyping technique via fluorescent PCR-capillary gel electrophoresis.First, cells are transfected with plasmids expressing Cas9-GFP fusion protein and individual sgRNA. Two days later, GFP-positive cells are sorted and plated onto 10-cm dishes. When individual colonies of cells are visible, they are picked and arrayed on 96-well plates. When the arrayed cells reach ~80% confluence, they are lysed directly using Direct-Lyse buffer and the crude lysate is used to amplify the genomic region containing the expected indel site using fluorophore-labelled primers. The labelled amplicons are resolved via capillary gel electrophoresis and successful mutants are identified by shifts in fragment size with respect to wildtype fragment. Putative knock-out clones are expanded and validated via Sanger sequencing, quantitative RT-PCR and/or Western blot analysis.
Mentions: Current methods of genotyping engineered nuclease-mediated mutations are not optimal for high-throughput screening of individual knockout clones due to inherent limitations and/or high cost. We thus sought to determine whether fluorescent PCR coupled with capillary gel electrophoresis is able to accurately genotype mutant clones in a high-throughput and cost-effective manner and hence circumvent the abovementioned limitations (see Fig. 1 for schematic of protocol).

Bottom Line: Recent advances in the engineering of sequence-specific synthetic nucleases provide enormous opportunities for genetic manipulation of gene expression in order to study their cellular function in vivo.Here, we describe an efficient method of genotyping clonal CRISPR/Cas9-mediated mutants in a high-throughput manner involving the use of a direct lysis buffer to extract crude genomic DNA straight from cells in culture, and fluorescent PCR coupled with capillary gel electrophoresis.This technique also allows for genotyping of multiplexed gene targeting in a single clone.

View Article: PubMed Central - PubMed

Affiliation: Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore 169857.

ABSTRACT
Recent advances in the engineering of sequence-specific synthetic nucleases provide enormous opportunities for genetic manipulation of gene expression in order to study their cellular function in vivo. However, current genotyping methods to detect these programmable nuclease-induced insertion/deletion (indel) mutations in targeted human cells are not compatible for high-throughput screening of knockout clones due to inherent limitations and high cost. Here, we describe an efficient method of genotyping clonal CRISPR/Cas9-mediated mutants in a high-throughput manner involving the use of a direct lysis buffer to extract crude genomic DNA straight from cells in culture, and fluorescent PCR coupled with capillary gel electrophoresis. This technique also allows for genotyping of multiplexed gene targeting in a single clone. Overall, this time- and cost-saving technique is able to circumvent the limitations of current genotyping methods and support high-throughput screening of nuclease-induced mutants.

No MeSH data available.


Related in: MedlinePlus