BatchPrimer3: A free web application for allele specific (SBE and allele flanking) primer design for SNPs genotyping in molecular diagnostics: A bioinformatics study

Ali, Seenaa Kadhum; Al-Koofee, Dhafer A.F.

doi:10.1016/j.genrep.2019.100524

Cited by 7 publications

(6 citation statements)

References 13 publications

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“…Thirty-five target sites in the bHLH gene family with predicted off-target sites for both Cas12a and Cas9 were selected for further testing. Primers for amplifying on-target sequences and a selection of predicted off-target sequences were designed using BatchPrimer3 32 in DNA sequences surrounding the target and predicted off-target sites extracted from the ITAG4.0 tomato genome build using BEDtools.…”

Section: Methodsmentioning

confidence: 99%

Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells

Slaman,

Kottenhagen,

de Martines

et al. 2024

Sci Rep

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Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-throughput amplicon sequencing on protoplasts. Of the three tested variants, Lachnospiraceae (Lb) Cas12a was the most efficient. Additionally, we developed an easy and effective Golden-Gate-based system for crRNA cloning. We compared LbCas12a to SpCas9 by investigating on-target efficacy and specificity at 35 overlapping target sites and 57 (LbCas12a) or 100 (SpCas9) predicted off-target sites. We found LbCas12a an efficient, robust addition to SpCas9, with similar overall though target-dependent efficiencies. LbCas12a induced more and larger deletions than SpCas9, which can be advantageous for specific genome editing applications. Off-target activity for LbCas12a was found at 10 out of 57 investigated sites. One or two mismatches were present distal from the PAM in all cases. We conclude that Cas12a-mediated genome editing is generally precise as long as such off-target sites can be avoided. In conclusion, we have determined the mutation pattern and efficacy of Cas12a-mediated CRISPR mutagenesis in tomato and developed a cloning system for the routine application of Cas12a for tomato genome editing.

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Section: Methodsmentioning

confidence: 99%

Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells

Slaman,

Kottenhagen,

de Martines

et al. 2024

Sci Rep

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“…PCN per chromosome was determined through real-time PCR method based on Škulj, et al (2008) 36 using the single-copy rpoD gene and the bla gene in pS4 as amplification targets. Primers for qPCR were designed with BatchPrimer3 62 with the following criteria: product size = 100 - 125 bp, primer size = 20 - 24 bp, primer Tm = 58 - 62 °C, primer GC % = 45-55 %. Cells were harvested at stationary phase (18 – 24 h) by centrifugation at 4500 rpm, 4 °C for 10 min and resuspended to OD 600 = 1 in cold PBS (1x, pH = 7.3).…”

Section: Methodsmentioning

confidence: 99%

Revealing the chassis-effect on a broad-host-range genetic switch and its concordance with interspecies bacterial physiologies

Chan

Baldwin

Bernstein

2023

Preprint

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Broad-host-range synthetic biology is an emerging frontier that aims to expand our current engineerable domain of microbial hosts for biodesign applications. As more novel species are brought to model status, synthetic biologists are discovering that identically engineered genetic circuits can exhibit different performances depending on the organism it operates within, an observation referred to as the chassis-effect. It remains a major challenge to uncover which genome encoded and physiological biological determinants will underpin chassis effects that govern the performance of engineered genetic devices. In this study, we compared model and novel bacterial hosts to ask whether phylogenomic relatedness or similarity in host physiology is a better predictor of toggle switch performance. This was accomplished using comparative framework based on multivariate statistical approaches to systematically demonstrate the chassis-effect and characterize the performance dynamics of a genetic toggle switch operating within six Gammaproteobacteria. Our results solidify the notion that genetic devices are significantly impacted by host-context. Furthermore, we formally determined that hosts exhibiting more similar metrics of growth and molecular physiology also exhibit more similar toggle switch performance, indicating that specific bacterial physiology underpins measurable chassis effects. The result of this study contributes to the field of broad-host-range synthetic biology by lending increased predictive power to the implementation of genetic devices in less-established microbial hosts.

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“…High-effect SNPs identified by SnpEff were further explored for the marker development and QTL validation. A total of 13 high-effect SNPs were converted into Kompetitive allele-specific PCR (KASP) markers using BatchPrimer3 software ( Ali and Al-Koofee, 2019 ; Supplementary Table 1 ) and tested for the association with PRSV-W resistance in the F 2 population ( n = 118). KASP assays were performed in 10-μl reaction volumes containing 5 μl of 2X low rox master mix, 0.16 μl of each forward primers (10 μm), 0.41 μl of reverse primer, 2 μl of genomic DNA (50 ng/μl), and 2.27 μl of H2O.…”

Section: Methodsmentioning

confidence: 99%

Whole Genome Re-sequencing and Bulk Segregant Analysis Reveals Chromosomal Location for Papaya Ringspot Virus W Resistance in Squash

Shrestha

Michael

et al. 2022

Front. Plant Sci.

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Squash (Cucurbita moschata) is among the most important cucurbit crops grown worldwide. Plant pathogen, Papaya ringspot virus W (PRSV-W) causes significant yield loss in commercial squash production globally. The development of virus-resistant cultivars can complement integrated disease management and mitigate losses due to viral infections. However, the genetic loci and molecular markers linked to PRSV-W resistance that could facilitate marker-assisted selection (MAS) for accelerated cultivar development are unknown. In this study, quantitative trait loci (QTL), molecular markers, and candidate genes associated with PRSV-W resistance in squash were identified in an F2 population (n = 118) derived from a cross between Nigerian Local accession (resistant) and Butterbush cultivar (susceptible). Whole genome re-sequencing-based bulked segregant analysis (QTLseq method; n = 10 for each bulk) and non-parametric interval mapping were used to identify a major QTL associated with PRSV-W resistance on chromosome 9 (QtlPRSV-C09) (p < 0.05) of C. moschata. QtlPRSV-C09 extended from 785,532 to 5,093,314 bp and harbored 12,245 SNPs among which 94 were high-effect variants. To validate QtlPRSV-C09, 13 SNP markers were assayed as Kompetitive allele-specific PCR (KASP) markers in the F2 population and tested for the association with PRSV-W resistance. Among these, two KASP markers (Ch09_2080834 and Ch09_5023865-1) showed significant association with PRSV-W resistance (p < 0.05). The two SNPs were located within exons of putative disease-resistant genes encoding the clathrin assembly family and actin cytoskeleton-regulatory complex proteins, which are implicated in disease resistance across plant species. The findings of this study will facilitate MAS for PRSV-W resistance in squash and allow further understanding of the functional mechanisms underlying potyvirus resistance in Cucurbita species.

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BatchPrimer3: A free web application for allele specific (SBE and allele flanking) primer design for SNPs genotyping in molecular diagnostics: A bioinformatics study

Cited by 7 publications

References 13 publications

Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells

Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells

Revealing the chassis-effect on a broad-host-range genetic switch and its concordance with interspecies bacterial physiologies

Whole Genome Re-sequencing and Bulk Segregant Analysis Reveals Chromosomal Location for Papaya Ringspot Virus W Resistance in Squash

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