Structure and engineering of Brevibacillus laterosporus Cas9.

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an unexpected architecture containing a triple-helix, which is specifically recognized by BlCas9, and that BlCas9 recognizes a unique N4CNDN protospacer adjacent motif through base-specific interactions on both the target and non-target DNA strands. Based on the structure, we rationally engineered a BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target scope in human cells. This approach may further improve the performance of the enhanced BlCas9 variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can be packaged into a single AAV vector for in vivo gene therapy.

Heart failure promotes multimorbidity through innate immune memory.

Patients with heart failure (HF) often experience repeated acute decompensation and develop comorbidities such as chronic kidney disease and frailty syndrome. Although this suggests pathological interaction among comorbidities, the mechanisms linking them are poorly understood. Here, we identified alterations in hematopoietic stem cells (HSCs) as a critical driver of recurrent HF and associated comorbidities. Bone marrow transplantation from HF-experienced mice resulted in spontaneous cardiac dysfunction and fibrosis in recipient mice, as well as increased vulnerability to kidney and skeletal muscle insults. HF enhanced the capacity of HSCs to generate proinflammatory macrophages. In HF mice, global chromatin accessibility analysis and single-cell RNA-seq showed that transforming growth factor-ß (TGF-ß) signaling was suppressed in HSCs, which corresponded with repressed sympathetic nervous activity in bone marrow. Transplantation of bone marrow from mice in which TGF-ß signaling was inhibited similarly exacerbated cardiac dysfunction. Collectively, these results suggest that cardiac stress modulates the epigenome of HSCs, which in turn alters their capacity to generate cardiac macrophage subpopulations. This change in HSCs may be a common driver of repeated HF events and comorbidity by serving as a key carrier of "stress memory."

Pooled CRISPR screening of high-content cellular phenotypes using ghost cytometry.

Recent advancements in image-based pooled CRISPR screening have facilitated the mapping of diverse genotype-phenotype associations within mammalian cells. However, the rapid enrichment of cells based on morphological information continues to pose a challenge, constraining the capacity for large-scale gene perturbation screening across diverse high-content cellular phenotypes. In this study, we demonstrate the applicability of multimodal ghost cytometry-based cell sorting, including both fluorescent and label-free high-content phenotypes, for rapid pooled CRISPR screening within vast cell populations. Using the high-content cell sorter operating in fluorescence mode, we successfully executed kinase-specific CRISPR screening targeting genes influencing the nuclear translocation of RelA. Furthermore, using the multiparametric, label-free mode, we performed large-scale screening to identify genes involved in macrophage polarization. Notably, the label-free platform can enrich target phenotypes without requiring invasive staining, preserving untouched cells for downstream assays and expanding the potential for screening cellular phenotypes even when suitable markers are absent.

An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.

DNA-GPS: A theoretical framework for optics-free spatial genomics and synthesis of current methods.

While single-cell sequencing technologies provide unprecedented insights into genomic profiles at the cellular level, they lose the spatial context of cells. Over the past decade, diverse spatial transcriptomics and multi-omics technologies have been developed to analyze molecular profiles of tissues. In this article, we categorize current spatial genomics technologies into three classes: optical imaging, positional indexing, and mathematical cartography. We discuss trade-offs in resolution and scale, identify limitations, and highlight synergies between existing single-cell and spatial genomics methods. Further, we propose DNA-GPS (global positioning system), a theoretical framework for large-scale optics-free spatial genomics that combines ideas from mathematical cartography and positional indexing. DNA-GPS has the potential to achieve scalable spatial genomics for multiple measurement modalities, and by eliminating the need for optical measurement, it has the potential to position cells in three-dimensions (3D).

Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range.

The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.

Deep distributed computing to reconstruct extremely large lineage trees.

Phylogeny estimation (the reconstruction of evolutionary trees) has recently been applied to CRISPR-based cell lineage tracing, allowing the developmental history of an individual tissue or organism to be inferred from a large number of mutated sequences in somatic cells. However, current computational methods are not able to construct phylogenetic trees from extremely large numbers of input sequences. Here, we present a deep distributed computing framework to comprehensively trace accurate large lineages (FRACTAL) that substantially enhances the scalability of current lineage estimation software tools. FRACTAL first reconstructs only an upstream lineage of the input sequences and recursively iterates the same produce for its downstream lineages using independent computing nodes. We demonstrate the utility of FRACTAL by reconstructing lineages from >235 million simulated sequences and from >16 million cells from a simulated experiment with a CRISPR system that accumulates mutations during cell proliferation. We also successfully applied FRACTAL to evolutionary tree reconstructions and to an experiment using error-prone PCR (EP-PCR) for large-scale sequence diversification.

CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations.

Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

Highly Multiplexed Analysis of CRISPR Genome Editing Outcomes in Mammalian Cells.

CRISPR-Cas-based genome editing has enabled efficient genetic engineering of a range of organisms and sparked revolutions in many fields of biology. After Streptococcus pyogenes Cas9 was first demonstrated for mammalian genome editing, many CRISPR-associated (Cas) protein variants have been isolated from different species and adopted for genome editing. Furthermore, various effector domains have been fused to these Cas proteins to expand their genome-editing abilities. Although the number of genome-editing tools has been rapidly increasing, the throughput of cell-based characterization of new genome-editing tools remains limited. Here we describe a highly multiplexed genome editing and sequencing library preparation protocol that allows high-resolution analysis of mutation outcomes and frequencies induced by hundreds to thousands of different genome-editing reagents in mammalian cells. We have successful experiences of developing several key genome-editing tools using this protocol. The protocol also is designed to be compatible with robotic liquid handling systems for further scalability.

Base editors for simultaneous introduction of C-to-T and A-to-G mutations.

We describe base editors that combine both cytosine and adenine base-editing functions. A codon-optimized fusion of the cytosine deaminase PmCDA1, the adenosine deaminase TadA and a Cas9 nickase (Target-ACEmax) showed a high median simultaneous C-to-T and A-to-G editing activity at 47 genomic targets. On-target as well as DNA and RNA off-target activities of Target-ACEmax were similar to those of existing single-function base editors.

Publisher Correction: Base editors for simultaneous introduction of C-to-T and A-to-G mutations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Complete Genome Sequence of Bacillus sp. Strain KH172YL63, Isolated from Deep-Sea Sediment.

Bacillus sp. strain KH172YL63 is a Gram-positive bacterium isolated from the deep-sea floor surface sediment at 3,308 m below sea level in the Nankai Trough in Japan. Here, we report the complete genome sequence of Bacillus sp. strain KH172YL63, which has a genome size of 4,251,700 bp and a G+C content of 44.8%.

DNA event recorders send past information of cells to the time of observation.

While current omics and single cell technologies have enabled measurements of high-resolution molecular snapshots of cells at a large scale, these technologies all require destruction of samples and prevent us from analyzing dynamic changes in molecular profiles, phenotypes, and behaviors of individual cells in a complex system. One possible direction to overcome this issue is the development of a cell-embedded 'event recorder' system, whereby molecular and phenotypic information of a cell(s) can be obtained at the time of observation with their past event information stored in 'heritable polymers' of the same cell. This concept has been demonstrated by many synthetic cellular circuits that monitor and transmit a certain set of environmental and intracellular signals into DNA, and have now been further accelerated by recent CRISPR-related technologies. Notably, the discovery of the RT-Cas1-Cas2 system, which acquires sequences of cellular transcripts into a specific host genomic region, has enabled recording of a broader range of molecular profile histories in the DNA tapes of cells, to understand the dynamics of complex biological processes that cannot be addressed by current technologies.

Complete Genome Sequence of Psychrobacter sp. Strain KH172YL61, Isolated from Deep-Sea Sediments in the Nankai Trough, Japan.

Psychrobacter sp. strain KH172YL61 is a Gram-negative bacterium isolated from deep-sea sediment in the Nankai Trough in Japan. Here, we report the complete genome sequence of this strain, which has a genome size of 3.19 Mb, with a G+C content of 44.0%.

Fast and global detection of periodic sequence repeats in large genomic resources.

Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. We developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. With a simple constraint, sequence periodicity, SPADE captured reported genome-editing-associated sequences and other protein families involving repeating domains such as tetratricopeptide, ankyrin and WD40 repeats with better performance than the other software designed for limited sets of repetitive biomolecular sequences, suggesting the high potential of this software to contribute to the discovery of new biological events and new genome editing modules.

Engineered CRISPR-Cas9 nuclease with expanded targeting space.

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

High-Quality Overlapping Paired-End Reads for the Detection of A-to-I Editing on Small RNA.

Paired-end RNA sequencing (RNA-seq) is usually applied to the quantification of long transcripts such as messenger or long non-coding RNAs, in which case overlapping pairs are discarded. In contrast, RNA-seq on short RNAs (≤ 200 nt) is typically carried out in single-end mode, as the additional cost associated with paired-end would only translate into redundant sequence information. Here, we exploit paired-end sequencing of short RNAs as a strategy to filter out sequencing errors and apply this method to the identification of adenosine-to-inosine (A-to-I) RNA editing events on human precursor microRNA (pre-miRNA) and mature miRNA. Combined with RNA immunoprecipitation sequencing (RIP-seq) of A-to-I RNA editing enzymes, this method takes full advantage of deep sequencing technology to identify RNA editing sites with unprecedented resolution in terms of editing efficiency.

Base-pairing probability in the microRNA stem region affects the binding and editing specificity of human A-to-I editing enzymes ADAR1-p110 and ADAR2.

Adenosine deaminases acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I). A-to-I RNA editing targets double-stranded RNA (dsRNA), and increases the complexity of gene regulation by modulating base pairing-dependent processes such as splicing, translation, and microRNA (miRNA)-mediated gene silencing. This study investigates the genome-wide binding preferences of the nuclear constitutive isoforms ADAR1-p110 and ADAR2 on human miRNA species by RNA immunoprecipitation of ADAR-bound small RNAs (RIP-seq). Our results suggest that secondary structure predicted by base-pairing probability in the mainly double-stranded region of a pre-miRNA or mature miRNA duplex may determine ADAR isoform preference for binding distinct subpopulations of miRNAs. Furthermore, we identify 31 unique editing sites with statistical significance, 19 sites of which are novel editing sites. Editing sites are enriched in the seed region responsible for target recognition by miRNAs, and isoform-specific nucleotide motifs in the immediate vicinity and opposite of editing sites are consistent with previous studies, and further reveal that ADAR2 may edit A/C bulges more frequently than ADAR1-p110 in the context of miRNA.