Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR

Haeussler, Maximilian; Shi, Kai; Eckert, Hélène; Eschstruth, Alexis; Mianné, Joffrey; Renaud, Jean; Schneider‐Maunoury, Sylvie; Shkumatava, Alena; Teboul, Lydia; Kent, J. H.; Joly, Jean‐Stéphane; Concordet, Jean‐Paul

doi:10.1186/s13059-016-1012-2

Cited by 1,445 publications

(1,371 citation statements)

References 54 publications

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“…The higher efficiency of the sgRNA2 compared to sgRNA1 could be explained by the difference in the targeted sequence. Nucleotide composition of the sgRNA can influence the efficiency of mutagenesis as showed by the CRISPOR program that evaluates the guide activity using prediction algorithms against seven available data sets (Haeussler et al ., 2016). sgRNA2 could be considered more efficient than sgRNA1 in 5 of the 7 algorithms used.…”

Section: Discussionmentioning

confidence: 99%

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

Morineau

Bellec

Tellier

et al. 2017

Plant Biotechnology Journal

234

166

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In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta‐12‐desaturase (FAD2) genes was achieved by CRISPR‐Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss‐of‐function mutants revealed the importance of delta‐12‐desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding.

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Section: Discussionmentioning

confidence: 99%

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

Morineau

Bellec

Tellier

et al. 2017

Plant Biotechnology Journal

234

166

View full text Add to dashboard Cite

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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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“…Two guide RNA sequences were selected on exons 1 and 2 of the MuPAXX gene using the CRISPOR web tool (http://crispor.tefor.net/crispor.py). 24 gRNA-overlapping restriction enzyme (RE) diagnostic sites near the Cas9 cleavage site were selected to facilitate mutagenesis screening and subsequent mouse genotyping (Supplementary Figures S1A and B). Double-stranded DNA oligonucleotides corresponding to the selected guide RNA were cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (the generous gift of Feng Zhang, Addgene, Cambridge MA, USA, #42230) according to Zhang lab's recommendations.…”

Section: Paxxmentioning

confidence: 99%

PAXX and Xlf interplay revealed by impaired CNS development and immunodeficiency of double KO mice

et al. 2017

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The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf − / − PAXX −/− E18.5 embryos is severely affected with the block of B-and T-cell maturation at the stage of IgH and TCRβ gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development. Cell Death and Differentiation (2018) 25, 444-452; doi:10.1038/cdd.2017; published online 27 October 2017All living organisms are subjected to multitude sources of DNA damage during their lifespan, either as a result of external assault or endogenous physiological processes.1 Among endogenous sources of physiological DNAdsb is the somatic rearrangement of immunoglobulin (Ig) and TCR genes in B and T lymphocytes, respectively, during the diversification of the adaptive immune system through V(D)J recombination.2 DNA double-stranded breaks (DNAdsb) are considered the most toxic lesions. DNAdsbs are repaired by two main mechanisms: the homologous recombination (HR) in cycling cells, when a sister chromatid is available as DNA repair template, and the non-homologous end joining (NHEJ) during all phases of the cell cycle.NHEJ proceeds via the simple religation of DNA ends without the need for a repair template.3 Briefly, the NHEJ is composed of seven core factors comprising the Ku70/80/ DNA-PKcs (DNA-dependent protein kinase catalytic subunit) complex, which recognizes and protects the broken DNA ends, the Artemis endo/exonuclease, which participates, when needed, in processing the DNA ends and the XRCC4/ DNA-Ligase IV/Xlf complex, which ultimately reseals the DNA break. The critical function of the NHEJ apparatus in various aspects of higher eukaryote development has been extensively perceived in several animal and human pathological conditions. As emblematic examples, loss of function of either XRCC4 or DNA ligase IV results in embryonic lethality in mice 4,5 and mutations in Artemis or DNA-PKcs result in severe combined immunodeficiency conditions in both men and mice, owing to aborted V(D)J recombination. 6 In addition, defects in NHEJ results in genetic instability and the propensity to develop various types of cancers, notably leukemia and lymphomas. Recently, a new DNA repair factor, PAXX (PAralog of XRCC4 and Xlf, also known as C9orf142 or XLS), has been identified independently by three laboratories based on bioinformatic...

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Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR

Cited by 1,445 publications

References 54 publications

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

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

PAXX and Xlf interplay revealed by impaired CNS development and immunodeficiency of double KO mice

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