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Synthetic Type III-E CRISPR-Cas Effectors for Programmable RNA-targeting.
The recent discovery of the type III-E class of CRISPR-Cas effectors has reshaped our fundamental understanding of CRISPR-Cas evolution and classification. Type III-E effectors are composed of several Cas7-like domains and a single Cas11-like domain naturally fused together to create a single polypeptide capable of targeting and degrading RNA. Here we identified a novel type III-E-like effector composed of three Cas7 domains and a Cas1 domain which was not active but could be engineered into an active chimeric RNA-targeting Cas effector by domain additions and swaps from other type III-E effectors. The results reveal that various domains in type III-E effectors can be swapped for the equivalent domain from a different type III-E effector. Remarkably, the Cas1 domain located at the C-terminus of Cas7-1 could be swapped in place of the Cas11 domain located between the Cas7.1 and the Cas7.2 domains of DiCas7-11. The results reveal a new modality for engineering type III-E effectors from the blueprints found in nature.
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Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance.
The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.
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A Reversibly Induced CRISPRi System Targeting Photosystem II in the Cyanobacterium Synechocystis sp. PCC 6803.
The cyanobacterium Synechocystis sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, P rhaBAD-RSW, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited photosystem II activity and growth of Synechocystis 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased photosystem II content and activity. This reversibly induced CRISPRi system in Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.
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Conformational landscape of the type V-K CRISPR-associated transposon integration assembly.
CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.
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Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.
CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.
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A programmable hierarchical-responsive nanoCRISPR elicits robust activation of endogenous target to treat cancer.
Despite promising progress of cancer gene therapy made, these therapeutics were still limited by the diversity of gene sizes and types. CRISPR/dCas9 mediated activation of tumor endogenous gene has shown great potential to surmount hinders of genetic varieties during the process of cancer gene therapy. However, the blood interference along with complicated tumor extra/intracellular microenvironment substantially compromise the performance of CRISPR/dCas9-based therapeutics in vivo. Methods: In this study, we constructed a programmable hierarchical-responsive nanoCRISPR (PICASSO) that can achieve sequential responses to the multiple physiological barriers in vivo. The core-shell structure endows PICASSO with long blood circulation capacity and tumor target accumulation as well as efficient cellular uptake and lysosomal escape, leading to high-performance of CRISPR/dCas9-mediated gene activation, which favors the antitumor efficacy. Results: Owing to these properties, PICASSO facilitated CRISPR/dCas9 mediated efficient transcriptional activation of various types of endogenous gene, and long non-protein-coding genes (LncRNA) containing targets ranging in size from ~1 kb to ~2000 kb in tumor cells. Intravenous administration of PICASSO to the tumor-bearing mice can achieve effective transcriptional activation of therapeutic endogenous gene, resulting in remarkable CRISPR/dCas9-mediate tumor inhibition with minimal adverse effect. Conclusions: Taken together, these characteristics allow PICASSO to unleash the potential of CRISPR/dCas9-based therapeutics in oncological treatment. The study provides a simple and versatile strategy to break through the restriction of sizes and types against cancer by utilization of tumor endogenous gene.
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In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation.
Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner.
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Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.
The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.
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Mini and enhanced CRISPR activators for cancer therapies.
INTRODUCTION: The RNA-guided nuclease Cas9 can be used as a programmable transcription activator, but there is still room for improvement in its effectiveness in eukaryotes, and its potential in cancer genetic therapy has been poorly investigated. OBJECTIVES: We aim to construct optimized CRISPRa tools and detect their potential role in cancer therapy by screening 9aa-TAD. METHODS: We selected a range of transcriptional coactivators for programmable activation and analyzed their effects on the expression of multiple endogenous genes using Flow cytometry and qRT-PCR. In order to improve the activation capacity of the CRISPRa tool, we fused the coactivators with the efficient dCas9-VPR system to construct a new activation system. Utilize RNA-seq to assess the activation specificity of genome-wide. To evaluate the value of the newly constructed activation system in cancer gene therapy, we activated the expression of the tumor suppressor genes PER2 and ZNF382, and performed changes in cancer cell proliferation qRT-PCR and clonal formation analysis. RESULTS: In this study, we screened the NHR module from C. elegans, which demonstrated a high transcription activation capacity with a compact size compared to VP64. We successfully demonstrated its efficiency in activating endogenous genes in mammalian cells. Furthermore, we developed an enhanced fused variant called NHR-VP64-p65-Rta (NVPR), which showed even higher efficiency compared to the previously established VPR module, making it an effective CRISPRa tool. The dCas9-NVPR complex also exhibited high specificity on a genome-wide scale. Finally, we utilized the dCas9-NVPR tool to restore the expression of tumor suppressor genes PER2 and ZNF382, effectively inhibiting the malignant phenotype of cancer cells. CONCLUSION: We have successfully developed and demonstrated a breakthrough CRISPRa tool with promising implications for cancer genetic therapy. This innovation expands the range of available gene editing tools and further validates the immense potential of CRISPR-based approaches in precision medicine.
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Measurement and clinical interpretation of CRISPR off-targets.
CRISPR genetic therapies are revolutionizing the landscape of preclinical research and clinical studies, providing new potential routes for curative intervention for a range of previously untreatable diseases. As with any therapy, the therapeutic benefits and risks must be weighed against consideration of the disease threat. Genome-related adverse events are an inherent risk of CRISPR genetic therapies, including off-target edits. The perception that CRISPR therapies ought to have near-zero off-targets belies clinical medicine, therapy development and biology, which demonstrate that 'perfect' therapeutics do not exist. Given that not all genomic off-target events are equal, we provide a practical framework to evaluate and assess off-target safety based on the tools available today and ones that will be developed in the future. With the comprehensive information and assessment gathered using these guidelines, we aim to streamline the transition of CRISPR therapeutics from bench to bedside.
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