Summary
CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region ...that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.
Circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) is a sensitive and unbiased method for defining the genome-wide activity (on-target and off-target) of ...CRISPR-Cas9 nucleases by selective sequencing of nuclease-cleaved genomic DNA (gDNA). Here, we describe a detailed experimental and analytical protocol for CIRCLE-seq. The principle of our method is to generate a library of circularized gDNA with minimized numbers of free ends. Highly purified gDNA circles are treated with CRISPR-Cas9 ribonucleoprotein complexes, and nuclease-linearized DNA fragments are then ligated to adapters for high-throughput sequencing. The primary advantages of CIRCLE-seq as compared with other in vitro methods for defining genome-wide genome editing activity are (i) high enrichment for sequencing nuclease-cleaved gDNA/low background, enabling sensitive detection with low sequencing depth requirements; and (ii) the fact that paired-end reads can contain complete information on individual nuclease cleavage sites, enabling use of CIRCLE-seq in species without high-quality reference genomes. The entire protocol can be completed in 2 weeks, including time for gRNA cloning, sequence verification, in vitro transcription, library preparation, and sequencing.
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment-refractory cancer. Molecular stratification in pancreatic cancer remains rudimentary and does not yet inform clinical ...management or therapeutic development. Here, we construct a high-resolution molecular landscape of the cellular subtypes and spatial communities that compose PDAC using single-nucleus RNA sequencing and whole-transcriptome digital spatial profiling (DSP) of 43 primary PDAC tumor specimens that either received neoadjuvant therapy or were treatment naive. We uncovered recurrent expression programs across malignant cells and fibroblasts, including a newly identified neural-like progenitor malignant cell program that was enriched after chemotherapy and radiotherapy and associated with poor prognosis in independent cohorts. Integrating spatial and cellular profiles revealed three multicellular communities with distinct contributions from malignant, fibroblast and immune subtypes: classical, squamoid-basaloid and treatment enriched. Our refined molecular and cellular taxonomy can provide a framework for stratification in clinical trials and serve as a roadmap for therapeutic targeting of specific cellular phenotypes and multicellular interactions.
Precise DNA cleavage using CRISPR-SpRYgests Christie, Kathleen A; Guo, Jimmy A; Silverstein, Rachel A ...
Nature biotechnology,
03/2023, Volume:
41, Issue:
3
Journal Article
Peer reviewed
Open access
Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas ...wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.
With increasing attention on the essential roles of the tumour microenvironment in recent years, the nervous system has emerged as a novel and crucial facilitator of cancer growth. In this Review, we ...describe the foundational, translational, and clinical advances illustrating how nerves contribute to tumour proliferation, stress adaptation, immunomodulation, metastasis, electrical hyperactivity and seizures, and neuropathic pain. Collectively, this expanding knowledge base reveals multiple therapeutic avenues for cancer neuroscience that warrant further exploration in clinical studies. We discuss the available clinical data, including ongoing trials investigating novel agents targeting the tumour–nerve axis, and the therapeutic potential for repurposing existing neuroactive drugs as an anti-cancer approach, particularly in combination with established treatment regimens. Lastly, we discuss the clinical challenges of these treatment strategies and highlight unanswered questions and future directions in the burgeoning field of cancer neuroscience.
CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector
Gene drives are based on an active source of Cas9 nuclease in the germline that ...promotes super-Mendelian inheritance of the transgene by homology-directed repair ("homing"). Understanding whether CRISPR-induced off-target mutations are generated in
mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations' exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most treatment refractory and lethal malignancies. The diversity of endothelial cell (EC) lineages in the tumor microenvironment (TME) impacts ...the efficacy of antineoplastic therapies, which in turn remodel EC states and distributions. Here, we present a single-cell resolution framework of diverse EC lineages in the PDAC TME in the context of neoadjuvant chemotherapy, radiotherapy, and losartan. We analyzed a custom single-nucleus RNA-seq dataset derived from 37 primary PDAC specimens (18 untreated, 14 neoadjuvant FOLFIRINOX + chemoradiotherapy, 5 neoadjuvant FOLFIRINOX + chemoradiotherapy + losartan). A single-nucleus transcriptome analysis of 15,185 EC profiles revealed two state programs (ribosomal, cycling), four lineage programs (capillary, arterial, venous, lymphatic), and one program that did not overlap significantly with prior signatures but was enriched in pathways involved in vasculogenesis, stem-like state, response to wounding and hypoxia, and endothelial-to-mesenchymal transition (reactive EndMT). A bulk transcriptome analysis of two independent cohorts (
= 269 patients) revealed that the lymphatic and reactive EndMT lineage programs were significantly associated with poor clinical outcomes. While losartan and proton therapy were associated with reduced lymphatic ECs, these therapies also correlated with an increase in reactive EndMT. Thus, the development and inclusion of EndMT-inhibiting drugs (e.g., nintedanib) to a neoadjuvant chemoradiotherapy regimen featuring losartan and/or proton therapy may be most effective in depleting both lymphatic and reactive EndMT populations and potentially improving patient outcomes.
Pancreatic ductal adenocarcinoma (PDAC) is a treatment-refractory malignancy in urgent need of a molecular framework for guiding therapeutic strategies. Bulk transcriptomic efforts over the past ...decade have yielded two broad consensus subtypes: classical pancreatic/epithelial versus basal-like/squamous/quasi-mesenchymal. Although this binary classification enables prognostic stratification, it does not currently inform the administration of treatments uniquely sensitive to either subtype. Furthermore, bulk mRNA studies are challenged by distinguishing contributions from the neoplastic compartment versus other cell types in the microenvironment, which is accentuated in PDAC given that neoplastic cellularity can be low. The application of single-cell transcriptomics to pancreatic tumors has generally lagged behind other cancer types due in part to the difficulty of extracting high-quality RNA from enzymatically degradative tissue, but emerging studies have and will continue to shed light on intratumoral heterogeneity, malignant-stromal interactions, and subtle transcriptional programs previously obscured at the bulk level. In conjunction with insights provided by single-cell/nucleus dissociative techniques, spatially resolved technologies should also facilitate the contextualization of gene programs and inferred cell-cell interactions within the tumor architecture. Finally, given that patients often receive neoadjuvant chemotherapy and/or chemoradiotherapy even in resectable disease, deciphering the gene programs enriched in or induced by cytotoxic therapy will be crucial for developing insights into complementary treatments aimed at eradicating residual cancer cells. Taken together, single-cell and spatial technologies provide an unprecedented opportunity to refine the foundations laid by prior bulk molecular studies and significantly augment precision oncology efforts in pancreatic cancer.
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