DSDecode: A Web-Based Tool for Decoding of Sequencing Chromatograms for Genotyping of Targeted Mutations

Liu, Weizhi; Xie, Xianrong; Ma, Xingliang; Li, Jun; Chen, Jiehu; Liu, Yao-Guang

doi:10.1016/j.molp.2015.05.009

Cited by 215 publications

(157 citation statements)

References 8 publications

(13 reference statements)

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“…napus . In total, the sequencing results of 422 PCR amplicons were analyzed by the decoding website DSDecode (http://dsdecode.scgene.com/) 46 . Part of the results were further confirmed by TA cloning and sequencing.…”

Section: Resultsmentioning

confidence: 99%

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CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus

Yang

Tang

et al. 2017

Sci Rep

162

118

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CRISPR/Cas9 is a valuable tool for both basic and applied research that has been widely applied to different plant species. Nonetheless, a systematical assessment of the efficiency of this method is not available for the allotetraploid Brassica napus—an important oilseed crop. In this study, we examined the mutation efficiency of the CRISPR/Cas9 method for 12 genes and also determined the pattern, specificity and heritability of these gene modifications in B. napus. The average mutation frequency for a single-gene targeted sgRNA in the T0 generation is 65.3%. For paralogous genes located in conserved regions that were targeted by sgRNAs, we observed mutation frequencies that ranged from 27.6% to 96.6%. Homozygotes were readily found in T0 plants. A total of 48.2% of the gene mutations, including homozygotes, bi-alleles, and heterozygotes were stably inherited as classic Mendelian alleles in the next generation (T1) without any new mutations or reversions. Moreover, no mutation was found in the putative off-target sites among the examined T0 plants. Collectively, our results demonstrate that CRISPR/Cas9 is an efficient tool for creating targeted genome modifications at multiple loci that are stable and inheritable in B. napus. These findings open many doors for biotechnological applications in oilseed crops.

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Section: Resultsmentioning

confidence: 99%

“…The online tool DSDecode (http://dsdecode.scgene.com/) 46 was used for chromatogram decoding. Sequence files (xxx.abi) and the reference gene sequences were uploaded to the server where they were decoded.…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus

Yang

Tang

et al. 2017

Sci Rep

162

118

View full text Add to dashboard Cite

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“…The PCR amplicons spanning the target sites were either subjected to direct Sanger sequencing, or cloned into the pMD19‐T vector (Takara Biomedical Technology, Beijing, China) and followed by Sanger sequencing. The chromatogram files were analysed by online tools (Liu et al ., ).…”

Section: Methodsmentioning

confidence: 97%

High‐efficiency genome editing using a dmc1 promoter‐controlled CRISPR/Cas9 system in maize

Feng

Han

et al. 2018

Plant Biotechnology Journal

108

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SummaryPrevious studies revealed that the promoters for driving both Cas9 and sgRNAs are quite important for efficient genome editing by CRISPR/Cas9 in plants. Here, we report our results of targeted genome editing using the maize dmc1 gene promoter combined with the U3 promoter for Cas9 and sgRNA, respectively. Three loci in the maize genome were selected for targeting. The T0 plants regenerated were highly efficiently edited at the target sites with homozygous or bi‐allelic mutants accounting for about 66%. The mutations in T0 plants could be stably transmitted to the T1 generation, and new mutations could be generated in gametes or zygotes. Whole‐genome resequencing indicated that no off‐target mutations could be detected in the predicted loci with sequence similarity to the targeted site. Our results show that the dmc1 promoter‐controlled (DPC) CRISPR/Cas9 system is highly efficient in maize and provide further evidence that the optimization of the promoters used for the CRISPR/Cas9 system is important for enhancing the efficiency of targeted genome editing in plants. The evolutionary conservation of the dmc1 gene suggests its potential for use in other plant species.

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“…For mutation detection, genomic DNA extracted from mutant seedlings (all plant) were used for PCR. Then PCR products (sequence is in Supplementary Table S3) were identified by comparing the 19-bp gRNA target sequences (tagttatggaatatgctgc) to the rice reference genome (sequence is in Supplementary Table S3) (Liu et al, 2015). In the T 0 generation, we collected 20 hygromycin-resistant plants.…”

Section: Methodsmentioning

confidence: 99%

OsSAPK2 Confers Abscisic Acid Sensitivity and Tolerance to Drought Stress in Rice

et al. 2017

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SNF 1-RELATED PROTEIN KINASE 2 (SnRK2) is a family of plant-specific protein kinases which is the key regulator of hyper-osmotic stress signaling and abscisic acid (ABA)-dependent development in various plants. Among the rice subclass-I and -II SnRK2s, osmotic stress/ABA–activated protein kinase 2 (SAPK2) may be the primary mediator of ABA signaling. However, SAPK2 has not been comprehensively characterized. In this study, we elucidated the functional properties of SAPK2 using loss-of-function mutants produced with the CRISPR/Cas9 system. The SAPK2 expression level was strongly upregulated by drought, high-salinity, and polyethylene glycol (PEG) treatments. The sapk2 mutants exhibited an ABA-insensitive phenotype during the germination and post-germination stages, suggesting that SAPK2 had a pivotal role related to ABA-mediated seed dormancy. The sapk2 mutants were more sensitive to drought stress and reactive oxygen species (ROS) than the wild-type plants, indicating that SAPK2 was important for responses to drought conditions in rice. An additional investigation revealed that SAPK2 increased drought tolerance in the following two ways: (i) by reducing water loss via the accumulation of compatible solutes, promoting stomatal closure, and upregulating the expression levels of stress-response genes such as OsRab16b, OsRab21, OsbZIP23, OsLEA3, OsOREB1 and slow anion channel (SLAC)-associated genes such as OsSLAC1 and OsSLAC7; (ii) by inducing the expression of antioxidant enzyme genes to promote ROS-scavenging abilities that will ultimately decrease ROS damages. Moreover, we also observed that SAPK2 significantly increased the tolerance of rice plants to salt and PEG stresses. These findings imply that SAPK2 is a potential candidate gene for future crop improvement studies.

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DSDecode: A Web-Based Tool for Decoding of Sequencing Chromatograms for Genotyping of Targeted Mutations

Cited by 215 publications

References 8 publications

CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus

CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus

High‐efficiency genome editing using a dmc1 promoter‐controlled CRISPR/Cas9 system in maize

OsSAPK2 Confers Abscisic Acid Sensitivity and Tolerance to Drought Stress in Rice

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