Abstract
Key message
Two methods, PCR and amplicon labeling based, were developed and successfully applied to reliably detect CRISPR/Cas9 induced indels in rice.
Abstract
The use of CRISPR/Cas9 has emerged as a powerful nuclease-based genome editing tool in several model organisms including plants for mutagenesis by inducing precise gene editing through efficient double strand DNA breaks (DSBs) at the target site and subsequent error-prone non-homologous end joining (NHEJ) repair, leading to indel mutations. Different molecular methods including enzymatic mismatch cleavage (EMC), high-resolution melting curve analysis (HRMA) and conventional polymerase chain reaction (PCR) combined with ligation detection reaction (LDR) have been developed to quick identify CRISPR/Cas9 induced mutations. However, their intrinsic drawbacks limit their application in the identification of indel mutants in plants. Here we present two methods (one simple PCR based and the other amplicon labeling based) for effective and sensitive detection of CRISPR/Cas9 induced indels in rice. In PCR-based method, targets were amplified using two pairs of primers for each target locus and visualized on gel electrophoresis, while in amplicon labeling-based method, targets were amplified using tri-primers (with one a universal 6-FAM 5′-labelled) and detected by DNA capillary electrophoresis. Both methods can accurately define indel sizes down to ± 1 bp, and are amenable for high throughput analysis, therefore, will significantly facilitate the identification of indel mutants generated by CRISPR/Cas9 for further functional analysis and breeding in rice and other plants.
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Acknowledgements
This work was supported by grants from the China National Transgenic Plant Special Fund (2016ZX08012-002) and the Programme of Introducing Talents of Discipline to Universities (111 Project, B14016).
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299_2019_2392_MOESM1_ESM.pdf
Optimization of PCR-based method and its application in the detection of CRISPR/Cas9 induced mutations. Amplicons of SD1 and other targeted genes were analyzed on 2% agarose gel electrophoresis analysis. a Optimization of PCR-based method using CRISPR/Cas9 induced SD1 mutations. Lane M, Trans 2K DNA marker; Lane 1, NTC (negative control); Lanes 2-5, wild-type (Wt), SD1 mutants with 1 bp, 2 bp, and 4 bp deletion, respectively. b Optimization of PCR-based method using CRISPR/Cas9 induced SD1 mutations. Lane M, Trans 2K DNA marker; Lane 1, NTC; Lanes 2-3, wild-type (Wt), SD1 mutants with 4 bp deletion and 3 bp insertion, respectively. c The application of the developed PCR based method in the detection of CRISPR/Cas9 induced mutations in LOC_Os03g12030. Lane M, Trans 2K DNA marker; Lane 1, NTC; Lane 2, wild-type (Wt); Lane 3, LOC_Os03g12030 mutant with 1 bp deletion. Left panels represent amplicons analyzed on 2% agarose gel electrophoresis, and right panels show Sanger sequencing results of corresponding mutants identified. d The application of the developed PCR based method in the detection of CRISPR/Cas9 induced mutations in LOC_Os09g08130. Lane M, Trans 2K DNA marker; Lane 1, NTC; Lane 2, wild-type (Wt); Lane 3, LOC_Os09g08130 mutant with 2 bp deletion. Left panels represent amplicons analyzed on 2% agarose gel electrophoresis, and right panels show Sanger sequencing results of corresponding mutants identified. e The application of the developed PCR based method in the detection of CRISPR/Cas9 induced mutations in LOC_Os07g26460. Lane M, Trans 2K DNA marker; Lane 1, NTC; Lane 2, wild-type (Wt); Lane 3-4, LOC_Os09g08130 mutants with 2 bp deletion and 2 bp deletion plus 1 bp insertion, respectively. Left panels represent amplicons analyzed on 2% agarose gel electrophoresis, and right panels show Sanger sequencing results of corresponding mutants identified. (PDF 346 KB)
299_2019_2392_MOESM2_ESM.pdf
Detection of insertion mutations in a single SD1 allele induced by CRISPR/Cas9 using developed PCR based method. a Analysis of PCR products amplified using SD1 Po and Pi primers on 2% agarose gel electrophoresis. Lane M, Trans 2K DNA marker; lane 2, wild-type (Wt); lane 3-6, SD1 mutants with insertions of 1A, 1T, 1G and 5A, respectively. b Quantification of the relative band intensities of corresponding Po and Pi PCR products in panel a using ImageJ. c Sanger sequencing results of corresponding PCR products in panel a. (PDF 200 KB)
299_2019_2392_MOESM3_ESM.pdf
Detection of replacement mutations in a single SD1 allele induced by CRISPR/Cas9 using developed PCR based method. a Analysis of PCR products amplified using SD1 Po and Pi primers on 2% agarose gel electrophoresis. Lane M, Trans 2K DNA marker; lane 2, wild-type (Wt); lane 3-7, SD1 mutants with base replacement of T to A and G to A, respectively. b Quantification of the relative band intensities of corresponding Po and Pi PCR products in panel a using ImageJ. c Sanger sequencing results of corresponding PCR products in panel a. (PDF 214 KB)
299_2019_2392_MOESM4_ESM.pdf
Optimization of amplicon labelling based method with SD1 mutants and its application in detection of a single LOC_Os06g04420 mutant induced by CRISPR/Cas9. a The optimization of the amplicon labelling based method. Genomic DNA from wild-type, CRISPR/Cas9 induced SD1 mutants with 4 bp deletion, and 5 bp insertion, respectively, was used for the PCR amplification using tri-primers, respectively, and resulting fluorophore labelled amplicons were analyzed in a fragment analyzer. X-axis and Y-axis represent amplicon size in base pairs and relative fluorescence units (RFU), respectively. Wt, wild-type; d4-HM, 4 bp deletion homozygous mutant; i5-HM, 5 bp insertion homozygous mutant. b Sensitivity test of the developed amplicon labelling method. A serial dilution of mixed samples containing 20%:80%; 10%:90%; 1%:99% and 0.1%:99.9% ratios of CRISPR/Cas9 induced SD1 mutant (Mt) (5 bp insertion) genomic DNA to wild- type (Wt) genomic DNA were used. The height of detected fluorophore labelled amplicon peak increased for wild-type but decreased for mutant alleles as the ratio of Mt:Wt in the mixture decreased. Red and green stars denote Mt allele and Wt allele, respectively. c Detection of mutations in a single LOC_Os06g04420 allele induced by CRISPR/Cas9. Fragment analysis of the fluorophore labelled amplicon of targets amplified using LOC_Os06g04420 tri-primers in a fragment analyzer. d Sanger sequencing results of corresponding mutations identified in c. (PDF 101 KB)
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Biswas, S., Li, R., Yuan, Z. et al. Development of methods for effective identification of CRISPR/Cas9-induced indels in rice. Plant Cell Rep 38, 503–510 (2019). https://doi.org/10.1007/s00299-019-02392-3
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DOI: https://doi.org/10.1007/s00299-019-02392-3