Long-term expandable mouse and human-induced nephron progenitor cells enable kidney organoid maturation and modeling of plasticity and disease.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.

A screen for regeneration-associated silencer regulatory elements in zebrafish.

Regeneration involves gene expression changes explained in part by context-dependent recruitment of transcriptional activators to distal enhancers. Silencers that engage repressive transcriptional complexes are less studied than enhancers and more technically challenging to validate, but they potentially have profound biological importance for regeneration. Here, we identified candidate silencers through a screening process that examined the ability of DNA sequences to limit injury-induced gene expression in larval zebrafish after fin amputation. A short sequence (s1) on chromosome 5 near several genes that reduce expression during adult fin regeneration could suppress promoter activity in stable transgenic lines and diminish nearby gene expression in knockin lines. High-resolution analysis of chromatin organization identified physical associations of s1 with gene promoters occurring preferentially during fin regeneration, and genomic deletion of s1 elevated the expression of these genes after fin amputation. Our study provides methods to identify "tissue regeneration silencer elements" (TRSEs) with the potential to reduce unnecessary or deleterious gene expression during regeneration.

UBE2N is essential for maintenance of skin homeostasis and suppression of inflammation.

UBE2N, a Lys63-ubiquitin conjugating enzyme, plays critical roles in embryogenesis and immune system development and function. However, its roles in adult epithelial tissue homeostasis and pathogenesis are unclear. We generated conditional mouse models that deleted Ube2n in skin cells in a temporally and spatially controlled manner. We found that Ube2n-knockout (KO) in the adult skin keratinocytes induced a range of inflammatory skin defects characteristic of psoriatic and actinic keratosis. These included eczematous inflammation, epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration, as well as signs of edema and blistering. Single cell transcriptomic analyses and RT-qPCR showed that Ube2n KO keratinocytes expressed elevated myeloid cell chemo-attractants such as Cxcl1 and Cxcl2 and decreased the homeostatic T lymphocyte chemo-attractant, Ccl27a. Consistently, the infiltrating immune cells of Ube2n-KO skin were predominantly myeloid-derived cells including neutrophils and M1-like macrophages that were highly inflammatory, as indicated by expression of Il1ß and Il24. Pharmacological blockade of the IL-1 receptor associated kinases (IRAK1/4) alleviated eczema, epidermal and dermal thickening, and immune infiltration of the Ube2n mutant skin. Together, these findings highlight a key role of keratinocyte-UBE2N in maintenance of epidermal homeostasis and skin immunity and identify IRAK1/4 as potential therapeutic target for inflammatory skin disorders.

Skeletal muscle regeneration failure in ischemic-damaged limbs is associated with pro-inflammatory macrophages and premature differentiation of satellite cells.

BACKGROUND: Chronic limb-threatening ischemia (CLTI), a severe manifestation of peripheral arterial disease (PAD), is associated with a 1-year limb amputation rate of approximately 15-20% and substantial mortality. A key feature of CLTI is the compromised regenerative ability of skeletal muscle; however, the mechanisms responsible for this impairment are not yet fully understood. In this study, we aim to delineate pathological changes at both the cellular and transcriptomic levels, as well as in cell-cell signaling pathways, associated with compromised muscle regeneration in limb ischemia in both human tissue samples and murine models of CLTI. METHODS: We performed single-cell transcriptome analysis of ischemic and non-ischemic muscle from the same CLTI patients and from a murine model of CLTI. In both datasets, we analyzed gene expression changes in macrophage and muscle satellite cell (MuSC) populations as well as differential cell-cell signaling interactions and differentiation trajectories. RESULTS: Single-cell transcriptomic profiling and immunofluorescence analysis of CLTI patient skeletal muscle demonstrated that ischemic-damaged tissue displays a pro-inflammatory macrophage signature. Comparable results were observed in a murine CLTI model. Moreover, integrated analyses of both human and murine datasets revealed premature differentiation of MuSCs to be a key feature of failed muscle regeneration in the ischemic limb. Furthermore, in silico inferences of intercellular communication and in vitro assays highlight the importance of macrophage-MuSC signaling in ischemia induced muscle injuries. CONCLUSIONS: Collectively, our research provides the first single-cell transcriptome atlases of skeletal muscle from CLTI patients and a murine CLTI model, emphasizing the crucial role of macrophages and inflammation in regulating muscle regeneration in CLTI through interactions with MuSCs.

Decoding Heterogenous Single-cell Perturbation Responses.

Understanding diverse responses of individual cells to the same perturbation is central to many biological and biomedical problems. Current methods, however, do not precisely quantify the strength of perturbation responses and, more importantly, reveal new biological insights from heterogeneity in responses. Here we introduce the perturbation-response score (PS), based on constrained quadratic optimization, to quantify diverse perturbation responses at a single-cell level. Applied to single-cell transcriptomes of large-scale genetic perturbation datasets (e.g., Perturb-seq), PS outperforms existing methods for quantifying partial gene perturbation responses. In addition, PS presents two major advances. First, PS enables large-scale, single-cell-resolution dosage analysis of perturbation, without the need to titrate perturbation strength. By analyzing the dose-response patterns of over 2,000 essential genes in Perturb-seq, we identify two distinct patterns, depending on whether a moderate reduction in their expression induces strong downstream expression alterations. Second, PS identifies intrinsic and extrinsic biological determinants of perturbation responses. We demonstrate the application of PS in contexts such as T cell stimulation, latent HIV-1 expression, and pancreatic cell differentiation. Notably, PS unveiled a previously unrecognized, cell-type-specific role of coiled-coil domain containing 6 (CCDC6) in guiding liver and pancreatic lineage decisions, where CCDC6 knockouts drive the endoderm cell differentiation towards liver lineage, rather than pancreatic lineage. The PS approach provides an innovative method for dose-to-function analysis and will enable new biological discoveries from single-cell perturbation datasets.

CRISPR-Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames.

Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR-Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the 'dark' proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.

TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons.

Trimethylation of histone H3 lysine 9 (H3K9me3) is crucial for the regulation of gene repression and heterochromatin formation, cell-fate determination and organismal development1. H3K9me3 also provides an essential mechanism for silencing transposable elements1-4. However, previous studies have shown that canonical H3K9me3 readers (for example, HP1 (refs. 5-9) and MPP8 (refs. 10-12)) have limited roles in silencing endogenous retroviruses (ERVs), one of the main transposable element classes in the mammalian genome13. Here we report that trinucleotide-repeat-containing 18 (TNRC18), a poorly understood chromatin regulator, recognizes H3K9me3 to mediate the silencing of ERV class I (ERV1) elements such as LTR12 (ref. 14). Biochemical, biophysical and structural studies identified the carboxy-terminal bromo-adjacent homology (BAH) domain of TNRC18 (TNRC18(BAH)) as an H3K9me3-specific reader. Moreover, the amino-terminal segment of TNRC18 is a platform for the direct recruitment of co-repressors such as HDAC-Sin3-NCoR complexes, thus enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis that disrupts the TNRC18(BAH)-mediated H3K9me3 engagement caused neonatal death in mice and, in multiple mammalian cell models, led to derepressed expression of ERVs, which affected the landscape of cis-regulatory elements and, therefore, gene-expression programmes. Collectively, we describe a new H3K9me3-sensing and regulatory pathway that operates to epigenetically silence evolutionarily young ERVs and exert substantial effects on host genome integrity, transcriptomic regulation, immunity and development.

HiCAR: Analysis of Open Chromatin Associated Long-range Chromatin Interaction Using Low-Input Materials.

Cis-regulatory elements (cREs) and their long-range interactions are crucial for spatial-temporal gene regulation. While cREs can be characterized as accessible chromatin sequences, comprehensively identifying their spatial interactions remains a challenge. We recently developed a method, HiCAR (Hi-C on Accessible Regulatory DNA), which combines Tn5 transposase and chromatin proximity ligation to analyze open chromatin-anchored interactions in low-input cells. Application of HiCAR in human embryonic stem cells and lymphoblastoid cells reveals high-resolution chromatin contacts with efficiency comparable to in situ Hi-C across various distance ranges. Moreover, HiCAR was successfully applied to 30,000 primary human muscle stem cells, showcasing its potential for analyzing chromatin accessibility and looping in low-input primary cells and clinical samples. Here, we provide a detailed step-by-step protocol to perform the updated HiCAR experiments. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Tn5 Transposase Assembly Basic Protocol 2: HiCAR Library Preparation.

Crosstalk between RNA m6A and DNA methylation regulates transposable element chromatin activation and cell fate in human pluripotent stem cells.

Transposable elements (TEs) are parasitic DNA sequences accounting for over half of the human genome. Tight control of the repression and activation states of TEs is critical for genome integrity, development, immunity and diseases, including cancer. However, precisely how this regulation is achieved remains unclear. Here we develop a targeted proteomic proximity labeling approach to capture TE-associated proteins in human embryonic stem cells (hESCs). We find that the RNA N6-methyladenosine (m6A) reader, YTHDC2, occupies genomic loci of the primate-specific TE, LTR7/HERV-H, specifically through its interaction with m6A-modified HERV-H RNAs. Unexpectedly, YTHDC2 recruits the DNA 5-methylcytosine (5mC)-demethylase, TET1, to remove 5mC from LTR7/HERV-H and prevent epigenetic silencing. Functionally, the YTHDC2/LTR7 axis inhibits neural differentiation of hESCs. Our results reveal both an underappreciated crosstalk between RNA m6A and DNA 5mC, the most abundant regulatory modifications of RNA and DNA in eukaryotes, and the fact that in hESCs this interplay controls TE activity and cell fate.

Modeling kidney development, disease, and plasticity with clonal expandable nephron progenitor cells and nephron organoids.

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identifying novel genes associated with kidney development and disease. A rapid, efficient, and scalable organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.

Single-cell chromatin accessibility profiling reveals a self-renewing muscle satellite cell state.

A balance between self-renewal and differentiation is critical for the regenerative capacity of tissue-resident stem cells. In skeletal muscle, successful regeneration requires the orchestrated activation, proliferation, and differentiation of muscle satellite cells (MuSCs) that are normally quiescent. A subset of MuSCs undergoes self-renewal to replenish the stem cell pool, but the features that identify and define self-renewing MuSCs remain to be elucidated. Here, through single-cell chromatin accessibility analysis, we reveal the self-renewal versus differentiation trajectories of MuSCs over the course of regeneration in vivo. We identify Betaglycan as a unique marker of self-renewing MuSCs that can be purified and efficiently contributes to regeneration after transplantation. We also show that SMAD4 and downstream genes are genetically required for self-renewal in vivo by restricting differentiation. Our study unveils the identity and mechanisms of self-renewing MuSCs, while providing a key resource for comprehensive analysis of muscle regeneration.

Pro-inflammatory macrophages impair skeletal muscle regeneration in ischemic-damaged limbs by inducing precocious differentiation of satellite cells.

Chronic limb-threatening ischemia (CLTI), representing the end-stage of peripheral arterial disease (PAD), is associated with a one-year limb amputation rate of ∻15-20% and significant mortality. A key characteristic of CLTI is the failure of the innate regenerative capacity of skeletal muscle, though the underlying mechanisms remain unclear. Here, single-cell transcriptome analysis of ischemic and non-ischemic muscle from the same CLTI patients demonstrated that ischemic-damaged tissue is enriched with pro-inflammatory macrophages. Comparable results were also observed in a murine CLTI model. Importantly, integrated analyses of both human and murine data revealed premature differentiation of muscle satellite cells (MuSCs) in damaged tissue and indications of defects in intercellular signaling communication between MuSCs and their inflammatory niche. Collectively, our research provides the first single-cell transcriptome atlases of skeletal muscle from CLTI patients and murine models, emphasizing the crucial role of macrophages and inflammation in regulating muscle regeneration in CLTI through interactions with MuSCs.

Prolonged hypernutrition impairs TREM2-dependent efferocytosis to license chronic liver inflammation and NASH development.

Obesity-induced chronic liver inflammation is a hallmark of nonalcoholic steatohepatitis (NASH)-an aggressive form of nonalcoholic fatty liver disease. However, it remains unclear how such a low-grade, yet persistent, inflammation is sustained in the liver. Here, we show that the macrophage phagocytic receptor TREM2, induced by hepatocyte-derived sphingosine-1-phosphate, was required for efferocytosis of lipid-laden apoptotic hepatocytes and thereby maintained liver immune homeostasis. However, prolonged hypernutrition led to the production of proinflammatory cytokines TNF and IL-1ß in the liver to induce TREM2 shedding through ADAM17-dependent proteolytic cleavage. Loss of TREM2 resulted in aberrant accumulation of dying hepatocytes, thereby further augmenting proinflammatory cytokine production. This ultimately precipitated a vicious cycle that licensed chronic inflammation to drive simple steatosis transition to NASH. Therefore, impaired macrophage efferocytosis is a previously unrecognized key pathogenic event that enables chronic liver inflammation in obesity. Blocking TREM2 cleavage to restore efferocytosis may represent an effective strategy to treat NASH.

Maf family transcription factors are required for nutrient uptake in the mouse neonatal gut.

There are fundamental differences in how neonatal and adult intestines absorb nutrients. In adults, macromolecules are broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely on internalization of whole macromolecules and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors MAFB and c-MAF as markers of terminally differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4α and HNF4γ, master regulators of enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of macromolecular nutrient uptake. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number, in response to loss of Maf proteins. Finally, we show that loss of BLIMP1, a repressor of adult enterocyte genes, shows highly overlapping changes in gene expression and similar defects in macromolecular uptake. This work defines transcriptional regulators that are necessary for nutrient uptake in neonatal enterocytes.

Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

The Hippo pathway mediates Semaphorin signaling.

Semaphorins were originally identified as axonal guidance molecules, but they also control processes such as vascular development and tumorigenesis. The downstream signaling cascades of Semaphorins in these biological processes remain unclear. Here, we show that the class 3 Semaphorins (SEMA3s) activate the Hippo pathway to attenuate tissue growth, angiogenesis, and tumorigenesis. SEMA3B restoration in lung cancer cells with SEMA3B loss of heterozygosity suppresses cancer cell growth via activating the core Hippo kinases LATS1/2 (large tumor suppressor kinase 1/2). Furthermore, SEMA3 also acts through LATS1/2 to inhibit angiogenesis. We identified p190RhoGAPs as essential partners of the SEMA3A receptor PlexinA in Hippo regulation. Upon SEMA3 treatment, PlexinA interacts with the pseudo-guanosine triphosphatase (GTPase) domain of p190RhoGAP and simultaneously recruits RND GTPases to activate p190RhoGAP, which then stimulates LATS1/2. Disease-associated etiological factors, such as genetic lesions and oscillatory shear, diminish Hippo pathway regulation by SEMA3. Our study thus discovers a critical role of Hippo signaling in mediating SEMA3 physiological function.

Myoblast deactivation within engineered human skeletal muscle creates a transcriptionally heterogeneous population of quiescent satellite-like cells.

Satellite cells (SCs), the adult Pax7-expressing stem cells of skeletal muscle, are essential for muscle repair. However, in vitro investigations of SC function are challenging due to isolation-induced SC activation, loss of native quiescent state, and differentiation to myoblasts. In the present study, we optimized methods to deactivate in vitro expanded human myoblasts within a 3D culture environment of engineered human skeletal muscle tissues ("myobundles"). Immunostaining and gene expression analyses revealed that a fraction of myoblasts within myobundles adopted a quiescent phenotype (3D-SCs) characterized by increased Pax7 expression, cell cycle exit, and activation of Notch signaling. Similar to native SCs, 3D-SC quiescence is regulated by Notch and Wnt signaling while loss of quiescence and reactivation of 3D-SCs can be induced by growth factors including bFGF. Myobundle injury with a bee toxin, melittin, induces robust myofiber fragmentation, functional decline, and 3D-SC proliferation. By applying single cell RNA-sequencing (scRNA-seq), we discover the existence of two 3D-SC subpopulations (quiescent and activated), identify deactivation-associated gene signature using trajectory inference between 2D myoblasts and 3D-SCs, and characterize the transcriptomic changes within reactivated 3D-SCs in response to melittin-induced injury. These results demonstrate the ability of an in vitro engineered 3D human skeletal muscle environment to support the formation of a quiescent and heterogeneous SC population recapitulating several aspects of the native SC phenotype, and provide a platform for future studies of human muscle regeneration and disease-associated SC dysfunction.

HiCAR is a robust and sensitive method to analyze open-chromatin-associated genome organization.

The long-range interactions of cis-regulatory elements (cREs) play a central role in gene regulation. cREs can be characterized as accessible chromatin sequences. However, it remains technically challenging to comprehensively identify their spatial interactions. Here, we report a new method HiCAR (Hi-C on accessible regulatory DNA), which utilizes Tn5 transposase and chromatin proximity ligation, for the analysis of open-chromatin-anchored interactions with low-input cells. By applying HiCAR in human embryonic stem cells and lymphoblastoid cells, we demonstrate that HiCAR identifies high-resolution chromatin contacts with an efficiency comparable with that of in situ Hi-C over all distance ranges. Interestingly, we found that the "poised" gene promoters exhibit silencer-like function to repress the expression of distal genes via promoter-promoter interactions. Lastly, we applied HiCAR to 30,000 primary human muscle stem cells and demonstrated that HiCAR is capable of analyzing chromatin accessibility and looping using low-input primary cells and clinical samples.

Tumor-propagating side population cells are a dynamic subpopulation in undifferentiated pleomorphic sarcoma.

Sarcomas contain a subpopulation of tumor-propagating cells (TPCs) with enhanced tumor-initiating and self-renewal properties. However, it is unclear whether the TPC phenotype in sarcomas is stable or a dynamic cell state that can derive from non-TPCs. In this study, we utilized a mouse model of undifferentiated pleomorphic sarcoma (UPS) to trace the lineage relationship between sarcoma side population (SP) cells that are enriched for TPCs and non-SP cells. By cotransplanting SP and non-SP cells expressing different endogenous fluorescent reporters, we show that non-SP cells can give rise to SP cells with enhanced tumor-propagating potential in vivo. Lineage trajectory analysis using single-cell RNA sequencing from SP and non-SP cells supports the notion that non-SP cells can assume the SP cell phenotype de novo. To test the effect of eradicating SP cells on tumor growth and self-renewal, we generated mouse sarcomas in which the diphtheria toxin receptor is expressed in the SP cells and their progeny. Ablation of the SP population using diphtheria toxin did not impede tumor growth or self-renewal. Altogether, we show that the sarcoma SP represent a dynamic cell state and targeting TPCs alone is insufficient to eliminate tumor progression.

CRISPR-SE: a brute force search engine for CRISPR design.

CRISPR is a revolutionary genome-editing tool that has been broadly used and integrated within novel biotechnologies. A major component of existing CRISPR design tools is the search engines that find the off-targets up to a predefined number of mismatches. Many CRISPR design tools adapted sequence alignment tools as the search engines to speed up the process. These commonly used alignment tools include BLAST, BLAT, Bowtie, Bowtie2 and BWA. Alignment tools use heuristic algorithm to align large amount of sequences with high performance. However, due to the seed-and-extend algorithms implemented in the sequence alignment tools, these methods are likely to provide incomplete off-targets information for ultra-short sequences, such as 20-bp guide RNAs (gRNA). An incomplete list of off-targets sites may lead to erroneous CRISPR design. To address this problem, we derived four sets of gRNAs to evaluate the accuracy of existing search engines; further, we introduce a search engine, namely CRISPR-SE. CRISPR-SE is an accurate and fast search engine using a brute force approach. In CRISPR-SE, all gRNAs are virtually compared with query gRNA, therefore, the accuracies are guaranteed. We performed the accuracy benchmark with multiple search engines. The results show that as expected, alignment tools reported an incomplete and varied list of off-target sites. CRISPR-SE performs well in both accuracy and speed. CRISPR-SE will improve the quality of CRISPR design as an accurate high-performance search engine.