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Fast and sensitive detection of indels induced by precise gene targeting.

Yang Z, Steentoft C, Hauge C, Hansen L, Thomsen AL, Niola F, Vester-Christensen MB, Frödin M, Clausen H, Wandall HH, Bennett EP - Nucleic Acids Res. (2015)

Bottom Line: The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies.Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site.The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.

View Article: PubMed Central - PubMed

Affiliation: Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark.

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Schematic depiction of the IDAA strategy. (a) Precise gene targeting creates double-stranded breaks that through NHEJ introduce indels at the target site. (b) Tri-primer PCR of the target region accomplished by use of target specific primers (F/R) flanking the target site and a universal 5′-FAM labelled primer (FamF) specific for a 5′-overhang sequence attached to primer F. Tri-primer PCR results in FAM amplicon labelling. (c) Fluorescently labelled amplicons containing the indels are detected by fragment analysis. Axis represent fluorescence intensity (FI) and amplicon size in base pairs.
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Figure 1: Schematic depiction of the IDAA strategy. (a) Precise gene targeting creates double-stranded breaks that through NHEJ introduce indels at the target site. (b) Tri-primer PCR of the target region accomplished by use of target specific primers (F/R) flanking the target site and a universal 5′-FAM labelled primer (FamF) specific for a 5′-overhang sequence attached to primer F. Tri-primer PCR results in FAM amplicon labelling. (c) Fluorescently labelled amplicons containing the indels are detected by fragment analysis. Axis represent fluorescence intensity (FI) and amplicon size in base pairs.

Mentions: The amplicon labelling strategy for evaluation of PCR product sizes was originally introduced by Oetting et al. (18), using polyacrylamide gel electrophoresis to resolve larger size differences in products. The strategy was further improved by Schuelke (19) using capillary electrophoresis for resolving smaller size differences down to ±2 bp of microsatellite repeats and a similar strategy was also used by Mellersh et al. (20). Here, we built further on this strategy and advanced the detection resolution down to a single base pair size discrimination. We used a single-step tri-primer PCR setup with a universal 6-FAM 5′-labelled primer (FamF) designed to a specific extension of the forward target specific primer, which enables one-step fluorophore labelling of amplicons derived from any given target using a universal amplification condition (Figure 1a). In contrast to previous M13 based primer designs (18,19), we used a non-M13 based primer design (5′-AGCTGACCGGCAGCAAAATTG-3′) increased in sequence length (21 versus 18 bases) and G/C content (52% versus 50%) for 6-FAM labelling, which enables use of increased assay stringency conditions and results in high product quality with single peak generation. The tri-primer amplification assay was standardized and optimized for general use in detecting a broad range of targets, and optimal amplicon yields were obtained using 10:1:10 molar ratios of 5′-labelled forward primer:unlabelledforward primer:reverse primer (Figure 1b and Supplementary Table S1). More than 50 different gene targets in different species have been profiled using these assay conditions. The fluorophore labelled amplicons can easily be detected with great sensitivity and with accurate size determination down to ±1 bp using standard DNA fragment analysis by capillary electrophoresis methodology (13) (Figure 1c). Importantly, the method applied enabled unbiased amplicon size discrimination with crude cell lysates as template source (Figure 3 and Supplementary Figures S1 and S2). To demonstrate the applicability of the IDAA strategy in nuclease-based genome editing, we first demonstrate its use for evaluating cutting efficiencies of CRISPR/Cas9 targeting using four different gRNA designs (Figure 2a). We targeted the Cosmc gene (X-linked) in CHO cells, because CHO only has one Cosmc allele and we have a very reliable phenotypic screen for knockout of Cosmc function (15). We found consistent targeting efficiencies of total cell pools when using either IDAA or phenotypic screening 3d after transfection with the constructs (Figure 2b). The IDAA analysis of the total cell pools and Sanger sequenced single cell clones furthermore revealed that indels ranged from +1 to -13 with an apparent frequency of <1% to a few percent for most of them (Figure 2a and c). Notably the identity of the predominant +1 (approximate frequency 20% and 32% for gRNA1 and gRNA2 respectively) insertion was validated by Sanger and found to be a T/A base-pair insertion (Figure 2d).


Fast and sensitive detection of indels induced by precise gene targeting.

Yang Z, Steentoft C, Hauge C, Hansen L, Thomsen AL, Niola F, Vester-Christensen MB, Frödin M, Clausen H, Wandall HH, Bennett EP - Nucleic Acids Res. (2015)

Schematic depiction of the IDAA strategy. (a) Precise gene targeting creates double-stranded breaks that through NHEJ introduce indels at the target site. (b) Tri-primer PCR of the target region accomplished by use of target specific primers (F/R) flanking the target site and a universal 5′-FAM labelled primer (FamF) specific for a 5′-overhang sequence attached to primer F. Tri-primer PCR results in FAM amplicon labelling. (c) Fluorescently labelled amplicons containing the indels are detected by fragment analysis. Axis represent fluorescence intensity (FI) and amplicon size in base pairs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4482057&req=5

Figure 1: Schematic depiction of the IDAA strategy. (a) Precise gene targeting creates double-stranded breaks that through NHEJ introduce indels at the target site. (b) Tri-primer PCR of the target region accomplished by use of target specific primers (F/R) flanking the target site and a universal 5′-FAM labelled primer (FamF) specific for a 5′-overhang sequence attached to primer F. Tri-primer PCR results in FAM amplicon labelling. (c) Fluorescently labelled amplicons containing the indels are detected by fragment analysis. Axis represent fluorescence intensity (FI) and amplicon size in base pairs.
Mentions: The amplicon labelling strategy for evaluation of PCR product sizes was originally introduced by Oetting et al. (18), using polyacrylamide gel electrophoresis to resolve larger size differences in products. The strategy was further improved by Schuelke (19) using capillary electrophoresis for resolving smaller size differences down to ±2 bp of microsatellite repeats and a similar strategy was also used by Mellersh et al. (20). Here, we built further on this strategy and advanced the detection resolution down to a single base pair size discrimination. We used a single-step tri-primer PCR setup with a universal 6-FAM 5′-labelled primer (FamF) designed to a specific extension of the forward target specific primer, which enables one-step fluorophore labelling of amplicons derived from any given target using a universal amplification condition (Figure 1a). In contrast to previous M13 based primer designs (18,19), we used a non-M13 based primer design (5′-AGCTGACCGGCAGCAAAATTG-3′) increased in sequence length (21 versus 18 bases) and G/C content (52% versus 50%) for 6-FAM labelling, which enables use of increased assay stringency conditions and results in high product quality with single peak generation. The tri-primer amplification assay was standardized and optimized for general use in detecting a broad range of targets, and optimal amplicon yields were obtained using 10:1:10 molar ratios of 5′-labelled forward primer:unlabelledforward primer:reverse primer (Figure 1b and Supplementary Table S1). More than 50 different gene targets in different species have been profiled using these assay conditions. The fluorophore labelled amplicons can easily be detected with great sensitivity and with accurate size determination down to ±1 bp using standard DNA fragment analysis by capillary electrophoresis methodology (13) (Figure 1c). Importantly, the method applied enabled unbiased amplicon size discrimination with crude cell lysates as template source (Figure 3 and Supplementary Figures S1 and S2). To demonstrate the applicability of the IDAA strategy in nuclease-based genome editing, we first demonstrate its use for evaluating cutting efficiencies of CRISPR/Cas9 targeting using four different gRNA designs (Figure 2a). We targeted the Cosmc gene (X-linked) in CHO cells, because CHO only has one Cosmc allele and we have a very reliable phenotypic screen for knockout of Cosmc function (15). We found consistent targeting efficiencies of total cell pools when using either IDAA or phenotypic screening 3d after transfection with the constructs (Figure 2b). The IDAA analysis of the total cell pools and Sanger sequenced single cell clones furthermore revealed that indels ranged from +1 to -13 with an apparent frequency of <1% to a few percent for most of them (Figure 2a and c). Notably the identity of the predominant +1 (approximate frequency 20% and 32% for gRNA1 and gRNA2 respectively) insertion was validated by Sanger and found to be a T/A base-pair insertion (Figure 2d).

Bottom Line: The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies.Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site.The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.

View Article: PubMed Central - PubMed

Affiliation: Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark.

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Related in: MedlinePlus