A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish

Varshney, Gaurav K.; Chen, Zelin; Fan, Chunxin; Ledin, Johan; Burgess, Shawn M.

doi:10.1038/nprot.2016.141

Cited by 179 publications

(192 citation statements)

References 34 publications

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“…In addition, the method has low sensitivity for the detection of single nucleotide mutations in mutant plasmid samples (Figure 5e). Varshney et al (2016) uses fluorescence-based PCR for the detection of CRISPR/ Cas9-induced mutations in zebrafish. That method is highly sensitive and does not require the handling of post-PCR products, but it does utilize highly expensive equipment, such as an ABI 3130xl or 3730xl genetic analyser.…”

Section: Discussionmentioning

confidence: 99%

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

Peng

Wang

et al. 2018

The Plant Journal

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Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene-edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high-throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high-throughput quantitative real-time (qPCR)-based method. The qPCR-based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild-type and a gene-edited mutant. We showed that the qPCR-based method can accurately distinguish CRISPR/Cas9-induced mutants from the wild-type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR-based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T transgenic plants. In a 384-well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post-polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T transgenic plants, which will be widely used in the area of plant gene editing.

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Section: Discussionmentioning

confidence: 99%

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

Peng

Wang

et al. 2018

The Plant Journal

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“…For ARb, we designed sgRNAs targeting sequence ARb-A, 5'-GGGAAACATGTGTT-CTCTAC-3', and ARb-B, 5'-GGGGGAAAGAAGAACTCCAT-3' whose cut sites were separated by 21 bp. We generated each sgRNA using cloning-free sgRNA synthesis (39). For instance, to synthesize sgRNA targeting ARa-A (gARa-A) we annealed oligonucleotide-ARa-A (oligo-ARa-A), which contained the ARa-A target sequence, and oligo-2, a generic oligo that we used for all sgRNA synthesis reactions (see Table S2 for all oligo sequences).…”

Section: Generation Of Frameshift Alleles For Ara and Arb Using Crispmentioning

confidence: 99%

Modular genetic control of social status in a cichlid fish

Alward

Laud²,

Skalnik

et al. 2020

Preprint

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Competing interest:We have no competing interests. Author's contributions: Conceptualization, B.A.A., R.D.F., S.A.J.; Methodology, B.A.A, S.A.J, C.J.S.; Investigation, B.A.A., C.J.S., R.A.Y, V.L.; Writing -Original Draft, B.A.A.; Writing -Review & Editing, B.A.A., R.A.Y, S.A.J.; Supervision, B.A.A. AbstractSocial hierarchies are ubiquitous in social species, yet the mechanisms underlying social status are unclear. In the African cichlid fish Astatotilapia burtoni, males stratify along a dominance hierarchy that varies based on testes mass, coloration, and behavior. Using androgen receptor (AR) mutant A. burtoni generated using CRISPR/Cas9, we find that two AR genes control social dominance. ARb, but not ARa, is required for testes growth and bright coloration, while ARa, but not ARb, is required for the performance of reproductive behavior and aggressive displays. Neither receptor is required for attacking males. Analysis of AR double mutants revealed that either AR is sufficient for attacking males. Social status in A. burtoni males is modularly controlled by ARa and ARb, indicating that these genes have undergone subfunctionalization.One Sentence Summary: Genetic dissection of social dominance in a cichlid using CRISPR/Cas9 gene editing reveals dissociable roles for distinct androgen receptor genes. Main textSocial animals -such as humans, non-human primates, mice, and fish -often organize into hierarchies (1). Within these social hierarchies dominant and non-dominant individuals exist, differing markedly along multiple behavioral and physiological dimensions. Dominant individuals typically behave more aggressively and have more mating opportunities than non-dominant individuals. Higher-ranking animals also tend to have higher levels of sex steroid hormones such as androgens (e.g., testosterone) and estrogens and larger gonads. Despite these compelling patterns, the mechanistic basis of social status remains to be established. Understanding what controls social status is important for several reasons. For instance, the underlying mechanisms of social status likely are involved in an animal's ability to interpret and navigate their social

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“…Zebrafish husbandry and embryo staging were performed according to Kimmel (42). All CRISPR/Cas9 mutagenesis was performed as previously described (43). The CRISPR targets and primers used for mutation detection are listed in the CRISPRz database (44) https://research.nhgri.nih.gov/CRISPRz/).…”

Section: Zebrafish Husbandry and Embryologymentioning

confidence: 99%

A subset of SMN complex members have a specific role in tissue regeneration via ERBB pathway-mediated proliferation

Pei

Chen

Slevin

et al. 2019

Preprint

Self Cite

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Spinal Muscular Atrophy (SMA) is the most common genetic disease in childhood. SMA is generally caused by mutations in SMN1. The Survival of Motor Neurons (SMN) complex consists of SMN1, Gemins (2-8) and Strap/Unrip. We previously demonstrated smn1 and gemin5 inhibited tissue regeneration in zebrafish. Here we investigated each individual SMN complex member and identified gemin3 as another regeneration-essential gene. These three genes are likely pan-regenerative since they affect the regeneration of hair cells, liver and caudal fin. RNA-Seq and miRNA-Seq analyses reveal that smn1, gemin3, and gemin5 are linked to a common set of genetic pathways, including the tp53 and ErbB pathways. Additional studies indicated all three genes facilitate regeneration by inhibiting the ErbB pathway, thereby allowing cell proliferation in the injured neuromasts. This study provides a new understanding of the SMN complex and a potential etiology for SMA and potentially other rare unidentified genetic diseases with similar symptoms.

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A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish

Cited by 179 publications

References 34 publications

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

High‐throughput detection and screening of plants modified by gene editing using quantitative real‐time polymerase chain reaction

Modular genetic control of social status in a cichlid fish

A subset of SMN complex members have a specific role in tissue regeneration via ERBB pathway-mediated proliferation

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