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The tripartite ParABS system mediates chromosome segregation in a wide range of bacteria. Dimeric ParB was proposed to nucleate on parS sites and spread to neighboring DNA. However, how properly distributed ParB dimers further compact chromosomal DNA into a higher-order nucleoprotein complex for partitioning remains poorly understood. Here, using a single-molecule approach, we show that tens of Bacillus subtilis ParB (Spo0J) proteins can stochastically multimerize on and stably bind to nonspecific DNA. The introduction of CTP promotes the formation and diffusion of the multimeric ParB along DNA, offering an opportunity for ParB proteins to further forgather and cluster. Intriguingly, ParB multimers can recognize parS motifs and are more inclined to remain immobile on them. Importantly, the ParB multimer features distinct capabilities of not only bridging two independent DNA molecules but also mediating their transportation, both of which are enhanced by the presence of either CTP or parS in the DNA. These findings shed new light on ParB dynamics in self-multimerization and DNA organization and help to better comprehend the assembly of the ParB-DNA partition complex.

The nucleolus is a multi-functional nuclear body. To tease out the roles of nucleolar structure without resorting to multi-action drugs, we knocked down RNA polymerase I subunit RPA194 in HeLa cells by siRNA. Loss of RPA194 resulted in nucleolar structural segregation and effects on both nucleolus-proximal and distal nuclear components. The perinucleolar compartment was disrupted, centromere-nucleolus interactions were significantly reduced, and the intranuclear locations of specific genomic loci were altered. Moreover, Cajal bodies, distal from nucleoli, underwent morphological and compositional changes. To distinguish whether these global reorganizations are the results of nucleolar structural disruption or inhibition of ribosome synthesis, the pre-ribosomal RNA processing factor, UTP4, was also knocked down, which did not lead to nucleolar segregation, nor the intranuclear effects seen with RPA195A knockdown, demonstrating that they do not arise from a cessation of ribosome synthesis. These findings point to a commutative system that links nucleolar structure to the maintenance and spatial organization of certain nuclear bodies and genomic loci.

The fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations. When binding at on-target sites, the tBE is transformed to cleave off the dCDI domain and catalyses targeted deamination for precise base editing. After delivery into mice through a dual-adeno-associated virus (AAV) system, the tBE system created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a ~30-40% decrease in total cholesterol. The development of tBE establishes a highly specific base editing system and its in vivo efficacy has potential for therapeutic applications.

Ma et al. use the CRISPR-Sirius system to track the dynamics of genomic loci situated kilobases to megabases apart on a single chromosome throughout the cell cycle, providing insight into the compaction–relaxation dynamics of the interphase chromosome fiber concurrent with changes in the overall movements of loci in the 4D genome., In contrast to the well-studied condensation and folding of chromosomes during mitosis, their dynamics during interphase are less understood. We deployed a CRISPR-based DNA imaging system to track the dynamics of genomic loci situated kilobases to megabases apart on a single chromosome. Two distinct modes of dynamics were resolved: local movements as well as ones that might reflect translational movements of the entire domain within the nucleoplasmic space. The magnitude of both of these modes of movements increased from early to late G1, whereas the translational movements were reduced in early S phase. The local fluctuations decreased slightly in early S and more markedly in mid-late S. These newly observed movements and their cell cycle dependence suggest the existence of a hitherto unrecognized compaction–relaxation dynamic of the interphase chromosome fiber, operating concurrently with changes in the extent of overall movements of loci in the 4D genome.

Fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications but suffers from off-target (OT) mutations. Here, we applied a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. This step-by-step protocol describes the plasmid construction of tBE system, determination of genome/transcriptome-wide OT mutations and tBE-mediated base editing in vivo.

The fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations. When binding at on-target sites, the tBE is transformed to cleave off the dCDI domain and catalyses targeted deamination for precise base editing. After delivery into mice through a dual-adeno-associated virus (AAV) system, the tBE system created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a ~30-40% decrease in total cholesterol. The development of tBE establishes a highly specific base editing system and its in vivo efficacy has potential for therapeutic applications.

In shallow freshwater ecosystems, submerged macrophytes and algae usually compete for nutrients, light and space. Previous studies mainly focused on the growth effects (biomass, growth rate, photosynthetic activities) of algae by submerged macrophytes. The present study found that submerged macrophytes could also have morphological influences on green algae. We used the green alga Chlorella vulgaris and the photosynthetic competitor Ceratophyllum demersum in our experiments. Results revealed the following: (1) both direct contact and extracts of macrophytes could promote colony formation by C. vulgaris; (2) high biomass of macrophytes caused the formation of more and larger colonies; (3) evidence was found to support the hypothesis that competitive pressure promotes facultative rather than constitutive colony formation. This temporary defensive strategy might be the result of increased carbohydrate production and secretion induced by macrophyte competition. After removal of macrophytes, colony cells reverted to individual cells and the accumulated carbohydrates in the colony cells could provide sufficient energy for rapid development of the algae. Thus, the response of C. vulgaris morphology to macrophytes might represent an effective co-evolutionary or co-existence strategy.

Epigenetic modifications play an essential role in chromatin architecture and dynamics. The role of epigenetic modification in chromatin organization has been studied by Hi-C from population cells, but imaging techniques to study their correlation and regulation in single living cells are lacking. Here we develop a CRISPR-based EpiGo (Epigenetic perturbation induced Genome organization) system to track epigenetic modification-mediated relocation, interaction or reorganization of genomic regions in living cells. EpiGo-KRAB is sufficient to induce the relocation of genomic loci to HP1α condensates and trigger genomic interactions. EpiGo-KRAB also triggers the induction of H3K9me3 at large genomic regions, which decorate on the surface of HP1α condensates possibly driven by phase separation.

We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. Sensitivity of STRIDE was tested using specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used TUNEL assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions), and fragmentation of DNA in human spermatozoa. STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

How CRISPR Cas9–guide RNA complexes navigate the nucleus and interrogate the genome is not well understood. Ma et al. track these complexes in live cells and find that mutations in the guide seed region significantly reduced the complex’s target residence time, with a commensurate impairment of cleavage., The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)–associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9–guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as

Supplementary notes. (PDF 339 kb)

Clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA scaffolds have been adapted to carry multiple binding sites for fluorescent proteins to enhance brightness for live cell imaging of genomic loci. However, many of these modifications result in guide RNA instability and thus produce lower genome-labeling efficiency than anticipated. Here we introduce CRISPR-Sirius, based on octet arrays of aptamers conferring both enhanced guide RNA stability and brightness, and provide initial biological applications of this platform.

To the EditorSchaefer et al.1 (referred to as Study_1) recently presented the provocative conclusion that CRISPR-Cas9 nuclease can induce many unexpected off-target mutations across the genome that arise from the sites with poor homology to the gRNA. As Wilson et al.2 pointed out, however, the selection of a co-housed mouse as the control is insufficient to attribute the observed mutation differences between the CRISPR-treated mice and control mice. Therefore, the causes of these mutations need to be further investigated. In 2015, Iyer et al.3 (referred to as Study_2) used Cas9 and a pair of sgRNAs to mutate the Ar gene in vivo and off-target mutations were investigated by comparison the control mice and the offspring of the modified mice. After analyzing the whole genome sequencing (WGS) of the offspring and the control mice, they claimed that off-target mutations are rare from CRISPR-Cas9 engineering. Notably, their study only focused on indel off-target mutations. We re-analyzed the WGS data of these two studies and detected both single nucleotide variants (SNVs) and indel mutations.

We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. The sensitivity of STRIDE was tested using a specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used terminal deoxynucleotidyl transferase dUTP nick-end labeling assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions) and fragmentation of DNA in human spermatozoa. The STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

We describe a transcription activator-like effector (TALE)-based strategy, termed "TALEColor," for labeling specific repetitive DNA sequences in human chromosomes. We designed TALEs for the human telomeric repeat and fused them with any of numerous fluorescent proteins (FPs). Expression of these TALE-telomere-FP fusion proteins in human osteosarcoma's (U2OS) cells resulted in bright signals coincident with telomeres. We also designed TALEs for centromeric sequences unique to certain chromosomes, enabling us to localize specific human chromosomes in live cells. Meanwhile we generated TALE-FPs in vitro and used them as probes to detect telomeres in fixed cells. Using human cells with different average telomere lengths, we found that the TALEColor signals correlated positively with telomere length. In addition, suspension cells were followed by imaging flow cytometry to resolve cell populations with differing telomere lengths. These methods may have significant potential both for basic chromosome and genome research as well as in clinical applications.

HeLa cells engineered with the fluorescent ubiquitinylation-based cell cycle indicator are used to study the connection between nucleolar stress and cell cycle progression. The results demonstrate a feedforward mechanism that leads to G2 arrest and identify ATR and Chk1 as molecular agents of the requisite checkpoint., We report experiments on the connection between nucleolar stress and cell cycle progression, using HeLa cells engineered with the fluorescent ubiquitinylation-based cell cycle indicator. Nucleolar stress elicited by brief exposure of cells to a low concentration of actinomycin D that selectively inhibits rRNA synthesis had no effect on traverse of G1 or S, but stalled cells in very late interphase. Additional experiments revealed that a switch occurs during a specific temporal window during nucleolar stress and that the subsequent cell cycle arrest is not triggered simply by the stress-induced decline in the synthesis of rRNA or by a ribosome starvation phenomenon. Further experiments revealed that this nucleolus stress-induced cell cycle arrest involves the action of a G2 checkpoint mediated by the ataxia telangiectasia and Rad3-related protein (ATR)–checkpoint kinase 1 (Chk1) pathway. Based on analysis of the cell cycle stages at which this nucleolar stress effect is put into action, to become manifest later, our results demonstrate a feedforward mechanism that leads to G2 arrest and identify ATR and Chk1 as molecular agents of the requisite checkpoint.

A lack of techniques to image multiple genomic loci in living cells has limited our ability to investigate chromosome dynamics. Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins. We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.

A major form of the severe pediatric muscle disease myotonic dystrophy is caused by a CTG repeat expansion in the gene (dmpk) for the enzyme DM protein kinase 1. As a consequence, this mutant allele produces aberrant transcripts that have elongated tracts of CUG repeats. These RNAs appear to stall within the cell nucleus, congressing in small, discrete bodies situated away from their site of gene transcription. Here, clustered regularly interspaced short palindromic repeats (CRISPR)-based approach is deployed that reveals that these nuclear nanobodies are not static structures. When the transcription of the dmpk gene is halted by a targeted CRISPR-based inhibition method, the preexisting transcripts delocalize from the nuclear bodies, showing that they are continuously trafficking through and are not stalled there in an accretion mode. This report is an example of how a novel method can address a nanoscale dimension of molecular cell biology in the nucleus, one with direct medical relevance.

Nucleostemin (NS) is a protein concentrated in the nucleolus of most stem cells and also in many tumor cells, which has been implicated in cell-cycle progression owing to its ability to modulate p53. Depletion of NS causes G(1) cell-cycle arrest, but its overexpression does so as well. Recently, this paradox has been clarified. NS overexpression causes a sequestration of murine double minute 2 (MDM2), preventing the destruction of p53. A recent study has demonstrated that loss of NS promotes the interaction of L5 and L11 ribosomal proteins with MDM2 and, thus, also prevents p53 degradation. This new finding expands our understanding of the multiple modes of NS action and reinforces the concept that the nucleolus has key roles in cell-cycle progression.

Nucleostemin (NS) is expressed in the nucleoli of adult and embryonic stem cells and in many tumors and tumor-derived cell lines. In coimmunoprecipitation experiments, nucleostemin is recovered with the tumor suppressor p53, and more recently we have demonstrated that nucleostemin exerts its role in cell cycle progression via a p53-dependent pathway. Here, we report that in human osteosarcoma cells, nucleostemin interacts with nucleophosmin, a nucleolar protein believed to possess oncogenic potential. Nucleostemin (NS) and nucleophosmin (NPM) displayed an extremely high degree of colocalization in the granular component of the nucleolus during interphase, and both proteins associated with prenucleolar bodies in late mitosis before the reformation of nucleoli. Coimmunoprecipitation experiments revealed that NS and NPM co-reside in complexes, and yeast two-hybrid experiments confirmed that they are interactive proteins, revealing the NPM-interactive region to be the 46-amino acid N-terminal domain of NS. In bimolecular fluorescence complementation studies, bright nucleolar signals were observed, indicating that these two proteins directly interact in the nucleolus in vivo. These results support the notion that cell cycle regulatory proteins congress and interact in the nucleolus, adding to the emerging concept that this nuclear domain has functions beyond ribosome production.

Nucleostemin (NS) is a nucleolar protein expressed in adult and embryo-derived stem cells, transformed cell lines, and tumors. NS decreases when proliferating cells exit the cell cycle, but it is unknown how NS is controlled, and how it participates in cell growth regulation. Here, we show that NS is down-regulated by the tumor suppressor p14ARF and that NS knockdown elevates the level of tumor suppressor p53. NS knockdown led to G1 cell cycle arrest in p53-positive cells but not in cells in which p53 was genetically deficient or depleted by small interfering RNA knockdown. These results demonstrate that, in the cells investigated, the level of NS is regulated by p14ARF and the control of the G1/S transition by NS operates in a p53-dependent manner.

Knowledge of an individual protein's modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501-511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.

Although the CRISPR-Cas9 system has been widely used for genome editing, little is known about its dynamics in live cells such as the kinetics of sgRNA:Cas9 assembly and DNA recognition. By stably expressing both fluorescent dCas9 and sgRNAs that specifically targets on chromosome 3 in live human cells we monitored the assembly of sgRNA:dCas9 complexes and tracked its dynamics of interactions with the target gene. In the absence of dCas9 expression, the sgRNA was so unstable as to not be detectable. In the presence of dCas9 the sgRNA was primarily localized in the nucleus and displayed a half-life within 15 minutes estimated by actinomycin chase experiments. These results are in agreement with sgRNA stability and sgRNA:dCas9 complex formation being the limiting step for DNA targeting. We next monitored the dynamics of on-target dCas9:sgRNA complexes by fluorescence recovery after photobleaching (FRAP), employing different lengths of sgRNA seed sequence and found that the recovery times of dCas9:sgRNA complexes on the target were about 6, 12 and 60 min. for a 7mer, 8mer and 11mer, respectively. This suggests that the complexes having shorter sgRNA seeds exchange with the target at a higher rate, implying a weaker affinity. We also compared the PAM sequences AG vs. GG, with the same length of the sgRNA seed sequence. The fluorescence recovery times were the same (> 60 min.), indicating similar affinities to the target and that the reported lower cleavage efficiency of AG PAM is possibly due to its inefficient targeting specificity.Our use of dCas9 in these in vivo studies has allowed us to deconvolve the overall CRISPR cycle to extract the DNA binding step and further to facilitate the optimization of Cas9-based gene editing.

Knowledge of an individual protein's modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501-511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.

Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.

A rare form of human ACAT1 mRNA, containing the optional long 5′-untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomal trans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7 (Li, B. L., Li, X. L., Duan, Z. J., Lee, O., Lin, S., Ma, Z. M., Chang, C. C., Yang, X. Y., Park, J. P., Mohandas, T. K., Noll, W., Chan, L., and Chang, T. Y. (1999) J. Biol. Chem. 274, 11060–11071). To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5′-untranslated repeat. To produce the 56-kDa ACAT1, acat1 sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1ORF. The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.

Very low-level expression of hepatitis B virus (HBV) preS1 with the native-type N-terminus hampered the biochemical and functional studies on its myristoylation. In the present study, the fusion HBV preS1 with the native-type N-terminus and a His6-Tag fused to C-terminus (HBV preS1-HT) was highly expressed in Escherichia coli. This was due to an introduced mutation of the rare codon GGA found in the HBV preS1 to the codon preferred by E. coli, GGU. The protein was rapidly purified from bacterial lysate by Ni-IDA affinity chromatography. The experimental assays using 3H-labeled substrate demonstrate that the purified HBV preS1-HT can be effectively N-myristoylated by recombinant human protein N-myristoyltransferase (NMT) in vitro.