Dataset Information

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots.

ABSTRACT: As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean.

SUBMITTER: Cai Y

PROVIDER: S-EPMC4540462 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots.

Cai Yupeng Y Chen Li L Liu Xiujie X Sun Shi S Wu Cunxiang C Jiang Bingjun B Han Tianfu T Hou Wensheng W

PloS one 20150818 8

As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation ...[more]

PMID: 26284791

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Project description:BackgroundTraditional CRISPR/Cas9 systems that rely on U6 or U3 snRNA promoters (RNA polymerase III-dependent promoters) can only achieve constitutive gene editing in plants, hampering the functional analysis of specifically expressed genes. Ribozyme-mediated CRISPR/Cas9 systems increase the types of promoters which can be used to transcribe sgRNA. Therefore, such systems allow specific gene editing; for example, transcription of the artificial gene Ribozyme-sgRNA-Ribozyme (RGR) is initiated by an RNA polymerase II-dependent promoter. Genetic transformation is indispensable for editing plant genes. In certain plant species, including pyrethrum, genetic transformation remains challenging to do, limiting the functional verification of novel CRISPR/Cas9 systems. Thus, this study's aim was to develop a simple Agrobacterium rhizogenes-mediated hairy root transformation system to analyze the function of a ribozyme-mediated CRISPR/Cas9 system in pyrethrum.ResultsA hairy root transformation system for pyrethrum is described, with a mean transformation frequency of 7%. Transgenic hairy roots transformed with the pBI121 vector exhibited significantly increased beta-glucuronidase staining as a visual marker of transgene expression. Further, a ribozyme-based CRISPR/Cas9 vector was constructed to edit the TcEbFS gene, which catalyzes synthesis of the defense-related compound (E)-β-farnesene in pyrethrum. The vector was transferred into the hairy roots of pyrethrum and two stably transformed hairy root transgenic lines obtained. Editing of the TcEbFS gene in the hairy roots was evaluated by gene sequencing, demonstrating that both hairy root transgenic lines had DNA base loss at the editing target site. Gas chromatography-mass spectrometry showed that the (E)-β-farnesene content was significantly decreased in both hairy root transgenic lines compared with the empty vector control group. Altogether, these results show that RGR can be driven by the CaMV35S promoter to realize TcEbFS gene editing in pyrethrum hairy roots.ConclusionAn A. rhizogenes-mediated hairy root transformation and ribozyme-mediated CRISPR/Cas9 gene editing system in pyrethrum was established, thereby facilitating gene editing in specific organs or at a particular developmental stage in future pyrethrum research.

Project description:UnlabelledThe prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani.ImportanceLeishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this study, we have implemented CRISPR-Cas9 genome-editing technology in L. donovani. Both single- and dual-gRNA expression vectors were developed using a strong RNA polymerase I promoter and ribozymes. With this system, it was possible to generate loss-of-function insertion and deletion mutations and introduce drug selection markers and the GFP sequence precisely into the L. donovani genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of Leishmania genes. This study further revealed that double-stranded DNA breaks created by CRISPR-Cas9 were repaired by the homology-directed repair (HDR) pathway and microhomology-mediated end joining (MMEJ) in Leishmania.

Dataset Information

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots.

Publications

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots.

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