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The recently developed single-cell CRISPR screening techniques, independently termed Perturb-Seq, CRISP-seq, or CROP-seq, combine pooled CRISPR screening with single-cell RNA-seq to investigate functional CRISPR screening in a single-cell granularity. Here, we present MUSIC, an integrated pipeline for model-based understanding of single-cell CRISPR screening data. Comprehensive tests applied to all the publicly available data revealed that MUSIC accurately quantifies and prioritizes the individual gene perturbation effect on cell phenotypes with tolerance for the substantial noise that exists in such data analysis. MUSIC facilitates the single-cell CRISPR screening from three perspectives, i.e., prioritizing the gene perturbation effect as an overall perturbation effect, in a functional topic-specific way, and quantifying the relationships between different perturbations. In summary, MUSIC provides an effective and applicable solution to elucidate perturbation function and biologic circuits by a model-based quantitative analysis of single-cell-based CRISPR screening data., Single-cell CRISPR screening combines pooled CRISPR screening with scRNA-seq analysis to expand the resolution power of genetic screening. Here, the authors develop MUSIC, a computational pipeline for analyzing single-cell CRISPR screening data.

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.

A major challenge for effective application of CRISPR systems is to accurately predict the single guide RNA (sgRNA) on-target knockout efficacy and off-target profile, which would facilitate the optimized design of sgRNAs with high sensitivity and specificity. Here we present DeepCRISPR, a comprehensive computational platform to unify sgRNA on-target and off-target site prediction into one framework with deep learning, surpassing available state-of-the-art in silico tools. In addition, DeepCRISPR fully automates the identification of sequence and epigenetic features that may affect sgRNA knockout efficacy in a data-driven manner. DeepCRISPR is available at http://www.deepcrispr.net/. Electronic supplementary material The online version of this article (10.1186/s13059-018-1459-4) contains supplementary material, which is available to authorized users.

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

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.

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.

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.