MiR26a reverses enzalutamide resistance in a bone-tumor targeted system with an enhanced effect on bone metastatic CRPC.

Resistance to androgen receptor (AR) inhibitors, including enzalutamide (Enz), as well as bone metastasis, are major challenges for castration-resistant prostate cancer (CRPC) treatment. In this study, we identified that miR26a can restore Enz sensitivity and inhibit bone metastatic CRPC. To achieve the highest combination effect of miR26a and Enz, we developed a cancer-targeted nano-system (Bm@PT/Enz-miR26a) using bone marrow mesenchymal stem cell (BMSC) membrane and T140 peptide to co-deliver Enz and miR26a. The in vitro/in vivo results demonstrated that miR26a can reverse Enz resistance and synergistically shrink tumor growth, invasion, and metastasis (especially secondary metastasis) in both subcutaneous and bone metastatic CRPC mouse models. We also found that the EZH2/SFRP1/WNT5A axis may be involved in this role. These findings open new avenues for treating bone metastatic and Enz-resistant CRPC.

Mechanisms of sunitinib resistance in renal cell carcinoma and associated opportunities for therapeutics.

Sunitinib is the first-line drug for renal cell carcinoma (RCC) treatment. However, patients who received sunitinib treatment will ultimately develop drug resistance after 6-15 months, creating a huge obstacle to the current treatment of renal cell carcinoma. Therefore, it is urgent to clarify the mechanisms of sunitinib resistance and develop new strategies to overcome it. In this review, the mechanisms of sunitinib resistance in renal cell carcinoma have been summarized based on five topics: activation of bypass or alternative pathway, inadequate drug accumulation, tumour microenvironment, metabolic reprogramming and epigenetic regulation. Furthermore, present and potential biomarkers, as well as potential treatment strategies for overcoming sunitinib resistance in renal cell carcinoma, are also covered.

Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems.

CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.

Primed adaptation tolerates extensive structural and size variations of the CRISPR RNA guide in Haloarcula hispanica.

Recent studies on CRISPR adaptation revealed that priming is a major pathway of spacer acquisition, at least for the most prevalent type I systems. Priming is guided by a CRISPR RNA which fully/partially matches the invader DNA, but the plasticity of this RNA guide has not yet been characterized. In this study, we extensively modified the two conserved handles of a priming crRNA in Haloarcula hispanica, and altered the size of its central spacer part. Interestingly, priming is insusceptible to the full deletion of 3' handle, which seriously impaired crRNA stability and interference effects. With 3' handle deletion, further truncation of 5' handle revealed that its spacer-proximal 6 nucleotides could provide the least conserved sequence required for priming. Subsequent scanning mutation further identified critical nucleotides within 5' handle. Besides, priming was also shown to tolerate a wider size variation of the spacer part, compared to interference. These data collectively illustrate the high tolerance of priming to extensive structural/size variations of the crRNA guide, which highlights the structural flexibility of the crRNA-effector ribonucleoprotein complex. The observed high priming effectiveness suggests that primed adaptation promotes clearance of the fast-replicating and ever-evolving viral DNA, by rapidly and persistently multiplexing the interference pathway.

Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon.

Research on CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated protein) systems has led to the revolutionary CRISPR/Cas9 genome editing technique. However, for most archaea and half of bacteria, exploitation of their native CRISPR-Cas machineries may be more straightforward and convenient. In this study, we harnessed the native type I-B CRISPR-Cas system for precise genome editing in the polyploid haloarchaeon Haloarcula hispanica. After testing different designs, the editing tool was optimized to be a single plasmid that carries both the self-targeting mini-CRISPR and a 600-800 bp donor. Significantly, chromosomal modifications, such as gene deletion, gene tagging or single nucleotide substitution, were precisely introduced into the vast majority of the transformants. Moreover, we showed that simultaneous editing of two genomic loci could also be readily achieved by one step. In summary, our data demonstrate that the haloarchaeal CRISPR-Cas system can be harnessed for genome editing in this polyploid archaeon, and highlight the convenience and efficiency of the native CRISPR-based genome editing strategy.

The spacer size of I-B CRISPR is modulated by the terminal sequence of the protospacer.

Prokaryotes memorize invader information by incorporating alien DNA as spacers into CRISPR arrays. Although the spacer size has been suggested to be predefined by the architecture of the acquisition complex, there is usually an unexpected heterogeneity. Here, we explored the causes of this heterogeneity in Haloarcula hispanica I-B CRISPR. High-throughput sequencing following adaptation assays demonstrated significant size variation among 37 957 new spacers, which appeared to be sequence-dependent. Consistently, the third nucleotide at the spacer 3΄-end (PAM-distal end) showed an evident bias for cytosine and mutating this cytosine in the protospacer sequence could change the final spacer size. In addition, slippage of the 5΄-end (PAM-end), which contributed to most of the observed PAM (protospacer adjacent motif) inaccuracy, also tended to change the spacer size. We propose that both ends of the PAM-protospacer sequence should exhibit nucleotide selectivity (with different stringencies), which fine-tunes the structural ruler, to a certain extent, to specify the spacer size.

DNA motifs determining the accuracy of repeat duplication during CRISPR adaptation in Haloarcula hispanica.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) acquire new spacers to generate adaptive immunity in prokaryotes. During spacer integration, the leader-preceded repeat is always accurately duplicated, leading to speculations of a repeat-length ruler. Here in Haloarcula hispanica, we demonstrate that the accurate duplication of its 30-bp repeat requires two conserved mid-repeat motifs, AACCC and GTGGG. The AACCC motif was essential and needed to be ∼10 bp downstream from the leader-repeat junction site, where duplication consistently started. Interestingly, repeat duplication terminated sequence-independently and usually with a specific distance from the GTGGG motif, which seemingly served as an anchor site for a molecular ruler. Accordingly, altering the spacing between the two motifs led to an aberrant duplication size (29, 31, 32 or 33 bp). We propose the adaptation complex may recognize these mid-repeat elements to enable measuring the repeat DNA for spacer integration.