Genome-Wide Prediction of Highly Specific Guide RNA Spacers for CRISPR–Cas9-Mediated Genome Editing in Model Plants and Major Crops

Xie, Kabin; Zhang, Jian Wei; Yang, Yinong

doi:10.1093/mp/ssu009

Cited by 293 publications

(194 citation statements)

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“…The U3p:sgRNA1 and U3p:sgRNA2 constructs were made as described previously (24). The specific spacer sequences for gRNA3-gRNA8 (SI Appendix, Table S3) were selected using the CRISPR-PLANT database (www.genome.arizona.edu/crispr/) (38). The PTG genes and U3p:PTG constructs were generated as described in SI Appendix, SI Methods, Figs.…”

Section: Methodsmentioning

confidence: 99%

Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

Xie

Minkenberg

Yang

2015

Proc. Natl. Acad. Sci. U.S.A.

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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

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Section: Methodsmentioning

confidence: 99%

Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

Xie

Minkenberg

Yang

2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

1,186

1,149

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“…A similar candidate target was suggested by three CRISPR designing tools (CRISPOR, CRISPR-P, and CRISPR-PLANT; Lei et al, 2014;Xie et al, 2014;Haeussler et al, 2016) and was selected for mutagenesis. A protospacer was designed using the ATTG_protospacer and AAAC_protospacer primers (Supplemental Table 3), and the annealed primers were cloned into pEn-Chimera according to Fauser et al (2014).…”

Section: Crispr Mutagenesismentioning

confidence: 99%

The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content

et al. 2018

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Phosphate starvation-mediated induction of the HAD-type phosphatases PPsPase1 (AT1G73010) and PECP1 (AT1G17710) has been reported in Arabidopsis (Arabidopsis thaliana). However, little is known about their in vivo function or impact on plant responses to nutrient deficiency. The preferences of PPsPase1 and PECP1 for different substrates have been studied in vitro but require confirmation in planta. Here, we examined the in vivo function of both enzymes using a reverse genetics approach. We demonstrated that PPsPase1 and PECP1 affect plant phosphocholine and phosphoethanolamine content, but not the pyrophosphate-related phenotypes. These observations suggest that the enzymes play a similar role in planta related to the recycling of polar heads from membrane lipids that is triggered during phosphate starvation. Altering the expression of the genes encoding these enzymes had no effect on lipid composition, possibly due to compensation by other lipid recycling pathways triggered during phosphate starvation. Furthermore, our results indicated that PPsPase1 and PECP1 do not influence phosphate homeostasis, since the inactivation of these genes had no effect on phosphate content or on the induction of molecular markers related to phosphate starvation. A combination of transcriptomics and imaging analyses revealed that PPsPase1 and PECP1 display a highly dynamic expression pattern that closely mirrors the phosphate status. This temporal dynamism, combined with the wide range of induction levels, broad expression, and lack of a direct effect on Pi content and regulation, makes PPsPase1 and PECP1 useful molecular markers of the phosphate starvation response.Plant growth is highly sensitive to a lack of phosphate (Pi); hence, the application of Pi fertilizers has become standard practice for high-throughput crop production (Cordell et al., 2009). Most phosphorus in the soil is not available to plants, as it is combined with other minerals or parts of organic compounds (Bieleski, 1973;Raghothama and Karthikeyan, 2005). Only a small fraction of soluble Pi (usually present at a concentration of ,10 mM in the soil) can be taken up by plants.Although growth is not optimal under limiting conditions, plants can withstand changing Pi concentrations within heterogeneous soils or in the external nutrient supply that can lead to reprogramming of their metabolism and architecture (Hammond et al., 2003;Péret et al., 2011;Plaxton and Tran, 2011). Root architecture can be modified to facilitate Pi uptake by favoring the development of lateral roots (at the expense of primary root elongation in many plants including Arabidopsis), increasing the density and length of root hairs, and limiting the development of aerial parts (López-Bucio et al., 2002;Svistoonoff et al., 2007;Gruber et al., 2013). Pi uptake mechanisms are enhanced at the root/soil interface, particularly through the stimulation of Pi transport activity (Mudge et al., 2002;Shin et al., 2004;Nussaume et al., 2011; Ayadi et al., 2015). In parallel, mobilization of Pi sources fr...

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“…In contrast, several recent studies have shown high specificity of CRISPR/Cas9 in plants Gao and Zhao, 2014;Zhang et al, 2014). The issue of off targeting in plants still needs to be addressed systematically, but several approaches can minimize possible impacts, such as using a recently developed algorithm for plant genomes that selects CRISPR/Cas9 sgRNAs with the least predicted off targets (Xie et al, 2014). For reverse genetics studies, choosing a few sgRNAs against a locus that has nonoverlapping off targets can be used to generate multiple independent alleles in different regions of the gene, thereby ensuring that any observed phenotype is caused by mutations in the desired locus rather than by an off target.…”

mentioning

confidence: 99%

Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System

et al. 2014

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Genome-Wide Prediction of Highly Specific Guide RNA Spacers for CRISPR–Cas9-Mediated Genome Editing in Model Plants and Major Crops

Cited by 293 publications

References 10 publications

Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content

Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System

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