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Genome targeting with CRISPR/Cas9 is a popular method for introducing mutations and creating knock-out effects. However, limited information is currently available on the mutagenesis of essential genes. This study investigated the efficiency of CRISPR/Cas9 in targeting rice essential genes: the singleton TARGET OF RAPAMYCIN (OsTOR) and the three paralogs of the Sucrose non-fermenting-1 (SNF1)-related kinase 1 (OsSnRK1α), OsSnRK1αA, OsSnRK1αB and OsSnRK1αC. Strong activity of constitutively expressed CRISPR/Cas9 was effective in creating mutations in OsTOR and OsSnRK1α genes, but inducible CRISPR/Cas9 failed to generate detectable mutations. The rate of OsTOR mutagenesis was relatively lower and only the kinase domain of OsTOR could be targeted, while mutations in the HEAT region were unrecoverable. OsSnRK1α paralogs could be targeted at higher rates; however, sterility or early senescence was observed in >50% of the primary mutants. Additionally, OsSnRK1αB and OsSnRK1αC, which bear high sequence homologies, could be targeted simultaneously to generate double-mutants. Further, although limited types of mutations were found in the surviving mutants, the recovered lines displayed loss-of-function or knockdown tor or snrk1 phenotypes. Overall, our data show that mutations in these essential genes can be created by CRISPR/Cas9 to facilitate investigations on their roles in plant development and environmental response in rice.

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor. Our observations suggest a multi-layered genetic response to drive tumor recurrence and implicate PTP4A2 (protein tyrosine phosphatase 4A2) as a modulator of self-renewal, proliferation and tumorigenicity in recurrent GBM. Genetic perturbation or small-molecule inhibition of PTP4A2 acts through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and its downstream molecular players, exploiting a functional dependency on ROBO signaling. Because a pan-PTP4A inhibitor was limited by poor penetrance across the blood–brain barrier in vivo, we engineered a second-generation chimeric antigen receptor (CAR) T cell therapy against ROBO1, a cell surface receptor enriched across recurrent GBM specimens. A single dose of ROBO1-targeted CAR T cells doubled median survival in cell-line-derived xenograft (CDX) models of recurrent GBM. Moreover, in CDX models of adult lung-to-brain metastases and pediatric relapsed medulloblastoma, ROBO1 CAR T cells eradicated tumors in 50–100% of mice. Our study identifies a promising multi-targetable PTP4A–ROBO1 signaling axis that drives tumorigenicity in recurrent GBM, with potential in other malignant brain tumors.

Constitutive expression of Cas9 leads to a higher editing efficiency; however, it also increases the chances of off-target mutations. Thus, transient expression of Cas9 is a desirable approach to achieve higher targeting efficiency and to curb the off-target effects. It was previously shown that heat-inducible expression of Cas9 had an editing efficiency of 45% as compared to the strong constitutive expression, and the heat-shock induced mutations were inherited by the next generation. In this study, cold-inducible promoter, AtRD29a, was used for driving Cas9 expression, and evaluated for its editing efficiency upon cold-treatment on the GUS transgene loci. The expression analysis of Cas9 before and after cold-shock showed ~ 2.5x increase in the expression, which is ~1000x lower as that of constitutive expression of Cas9 by rice ubiquitin-1 promoter (OsUbi1). Further, the targeting efficiency of Cas9 expressed under constitutive (OsUbi1:Cas9), cold-shock (AtRD29a:Cas9) or heat-shock (HSP:Cas9) promoters was tested on rice Target of Rapamycin (OsTOR) gene. The OsTOR gene is known to regulate various anabolic processes such as cell cycle, ribosome biogenesis, and photosynthesis. The HEAT repeat regions and kinase domain region of OsTOR were targeted. Among 21 primary transgenic (T0) plants of OsUbi1:Cas9 representing 9 independent transgenic lines, HEAT site was found to be wild type (WT), while monoallelic or biallelic mutations were observed in the Kinase site in 4 plants representing 3 independent lines. The analysis of 33 T1 progeny studied from three T0 plants showed that 22 (66%) inherited the mutations at Kinase domain; but no de-novo mutations in HEAT site were observed. In HSP:Cas9 and AtRD29a:Cas9 lines, none of the 40 T0 plants, which represented 17 independent lines exhibited mutations in either site. The expression analysis of a subset of these lines, showed 2-11x and 2-43x induced transcript levels of Cas9 in HSP:Cas9 and AtRD29a:Cas9 lines, respectively, indicating proper regulation of the Cas9. Beside OsTOR gene, the targeting efficiency of AtRD29a-, HSP- and OsUbi1-CRISPR/Cas9 was tested on the OsPDS gene, the key enzyme in carotenoid biosynthesis pathway. The analysis of Cas9 positive samples in these lines showed only WT sequences in all plants except for one OsUbi1:Cas9, which harbored biallelic mutations, indicating overall lower targeting efficiency in this experiment. In summary, the expression of Cas9 under heat and cold inducible promoters was under proper regulation as it did not exhibit any mutations at room-temperature. However, more analysis is needed to determine whether cold-induced expression of Cas9 by AtRD29a promoter is sufficient to create targeted mutations in the rice genome.