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Although changes in alternative splicing have been observed in cancer, their functional contributions still remain largely unclear. Here we report that splice isoforms of the cancer stem cell (CSC) marker CD44 exhibit strikingly opposite functions in breast cancer. Bioinformatic annotation in patient breast cancer in The Cancer Genome Atlas (TCGA) database reveals that the CD44 standard splice isoform (CD44s) positively associates with the CSC gene signatures, whereas the CD44 variant splice isoforms (CD44v) exhibit an inverse association. We show that CD44s is the predominant isoform expressed in breast CSCs. Elimination of the CD44s isoform impairs CSC traits. Conversely, manipulating the splicing regulator ESRP1 to shift alternative splicing from CD44v to CD44s leads to an induction of CSC properties. We further demonstrate that CD44s activates the PDGFRβ/Stat3 cascade to promote CSC traits. These results reveal CD44 isoform specificity in CSC and non-CSC states and suggest that alternative splicing provides functional gene versatility that is essential for distinct cancer cell states and thus cancer phenotypes.

Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that d C as9-APEX2 B iotinylation at genomic E lements by R estricted S patial T agging (C-BERST) allows the unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres. C-BERST enables the high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology. Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that d C as9-APEX2 B iotinylation at genomic E lements by R estricted S patial T agging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. By combining the spatially restricted enzymatic tagging enabled by APEX2 with programmable DNA targeting by dCas9, C-BERST has successfully identified nearly 50% of known telomere-associated factors and many known centromere-associated factors. We also identified and validated SLX4IP and RPA3 as telomeric factors, confirming C-BERST9s utility as a discovery platform. C-BERST enables the rapid, high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology.

In recent years, attractive toxic sugar bait has been used in the mosquito control in nature, and achieved good control effects. However, the current researches about toxic sugar bait did not focus on whether the wild mosquito population used for control is resistant or not. The purpose of this study was to evaluate the effectiveness of the toxic sugar bait against mosquito resistant populations to test the effects of bait on the control of mosquitoes with different levels of resistance. Boric acid, dinotefuran and deltamethrin were separately formulated into toxic sugar bait to test their anti-mosquito activity against Culex quinquefasciatus. Using the sugar baits formulated with boric acid and dinotefuran, the mortality of Cx. quinquefasciatus resistant populations was significantly higher than that of sensitive populations at the same concentration. Conversely, with the use of sugar baits formulated with deltamethrin, the mortality of Cx. quinquefasciatus resistant populations was significantly lower than that of sensitive populations at the same concentration. The results suggested that toxic sugar baits might have a good application prospect in high resistant mosquito management.

Genome organization and subnuclear protein localization are essential for normal cellular function and have been implicated in the control of gene expression, DNA replication, and genomic stability. The coupling of chromatin conformation capture (3C), chromatin immunoprecipitation and sequencing, and related techniques have continuously improved our understanding of genome architecture. To profile site-specifically DNA-associated proteins in a high-throughput and unbiased manner, the RNA-programmable CRISPR–Cas9 platform has recently been combined with an enzymatic labeling system to allow proteomic landscapes at repetitive and nonrepetitive loci to be defined with unprecedented ease and resolution. In this chapter, we describe the dCas9-APEX2 experimental approach for specifically targeting a DNA sequence, enzymatically labeling local proteins with biotin, and quantitatively analyzing the labeled proteome. We also discuss the optimization and extension of this pipeline to facilitate its use in understanding nuclear and chromosome biology.

Background The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery. Results Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N4GATT-3′), for NmeCas9 genome editing in human cells. Conclusions Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering. Electronic supplementary material The online version of this article (10.1186/s13059-018-1591-1) contains supplementary material, which is available to authorized users.

CRISPR-Cas systems are widely used for genome engineering technologies, and in their natural setting, they play crucial roles in bacterial and archaeal adaptive immunity, protecting against phages and other mobile genetic elements. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems1. We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature2–4. We have identified new anti-CRISPRs using the bioinformatic approach that successfully identified previous Acr proteins1 against Neisseria meningitidis Cas9 (NmeCas9). In this work we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems5. We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect co-evolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.

The epithelial-mesenchymal transition (EMT) is a fundamental developmental process that is abnormally activated in cancer metastasis. Dynamic changes in alternative splicing occur during EMT. ESRP1 and hnRNPM are splicing regulators that promote an epithelial splicing program and a mesenchymal splicing program, respectively. The functional relationships between these splicing factors in the genome-scale remain elusive. Comparing alternative splicing targets of hnRNPM and ESRP1 revealed that they co-regulate a set of cassette exon events, with the majority showing discordant splicing regulation. hnRNPM discordantly regulated splicing events show a positive correlation with splicing during EMT while concordant splicing events do not, highlighting the antagonistic role of hnRNPM and ESRP1 during EMT. Motif enrichment analysis near co-regulated exons identifies guanine-uridine rich motifs downstream of hnRNPM-repressed and ESRP1-enhanced exons, supporting a model of competitive binding to these cis-elements to antagonize alternative splicing. The set of co-regulated exons are enriched in genes associated with cell-migration and cytoskeletal reorganization, which are pathways associated with EMT. Splicing levels of co-regulated exons are associated with breast cancer patient survival and correlate with gene sets involved in EMT and breast cancer subtypes. These data identify complex modes of interaction between hnRNPM and ESRP1 in regulation of splicing in disease-relevant contexts.

The epithelial–mesenchymal transition (EMT) is a fundamental developmental process that is abnormally activated in cancer metastasis. Dynamic changes in alternative splicing occur during EMT. ESRP1 and hnRNPM are splicing regulators that promote an epithelial splicing program and a mesenchymal splicing program, respectively. The functional relationships between these splicing factors in the genome scale remain elusive. Comparing alternative splicing targets of hnRNPM and ESRP1 revealed that they coregulate a set of cassette exon events, with the majority showing discordant splicing regulation. Discordant splicing events regulated by hnRNPM show a positive correlation with splicing during EMT; however, concordant events do not, indicating the role of hnRNPM in regulating alternative splicing during EMT is more complex than previously understood. Motif enrichment analysis near hnRNPM–ESRP1 coregulated exons identifies guanine–uridine rich motifs downstream from hnRNPM-repressed and ESRP1-enhanced exons, supporting a general model of competitive binding to these cis-elements to antagonize alternative splicing. The set of coregulated exons are enriched in genes associated with cell migration and cytoskeletal reorganization, which are pathways associated with EMT. Splicing levels of coregulated exons are associated with breast cancer patient survival and correlate with gene sets involved in EMT and breast cancer subtyping. This study identifies complex modes of interaction between hnRNPM and ESRP1 in regulation of splicing in disease-relevant contexts.

BackgroundThe development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wild-type SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large (~4.2 kb) open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs (e.g. from Staphylococcus aureus, Campylobacter jejuni, Geobacillus stearothermophilus and Neisseria meningitidis) are considerably smaller and therefore better suited for viral delivery.ResultsHere we show that wild-type NmeCas9, when programmed with guide sequences of natural length (24 nucleotides), exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5’-N4GATT-3’), for NmeCas9 genome editing in human cells.ConclusionsOur results show that NmeCas9 is a naturally high-fidelity genome editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial–mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFβ signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFβ-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.