Cas9 Activity Research Articles

CRISPR/Cas9-Mediated Epigenome Editing of the IL-10 Gene for Targeted Whole Organ Gene Therapy for Lung Transplant

<h3>Purpose</h3> Anti-inflammatory and immunomodulatory gene therapy has shown promise in lung transplant. CRISPR/Cas9 technology unleashed the possibility of making gene therapy persistent by radically altering gene expression through precise DNA targeting. We envisioned activation of the endogenous IL-10 gene, which codes for this anti-inflammatory cytokine, using epigenome editing via CRISPR/Cas9 in the lung. This study investigated the efficacy and specificity in vitro and in rat lungs. <h3>Methods</h3> Adenoviral vectors were developed expressing a Cas9 activator and either one or two gRNAs targeting the promoter region of the IL-10 gene in the genome (Fig. A). Lewis rats were divided into three groups (Fig. B, n=5-10), trans-bronchially administered adenovirus (1 × 10⁸ PFU/rat) under standard transplant triple immunosuppression, and assessed after 72 hours. For specificity, primary lung fibroblasts treated with adenovirus were analyzed at 48 hours using chromatin immunoprecipitation (ChIP) of Cas9 followed by qPCR designed against the on- and off-target regions. <h3>Results</h3> The two-gRNA group showed significantly increased IL-10 gene expression compared to the diluent group (relative expression: 19.0±10.8 vs 1.3±1.2, p=0.001) and the single gRNA group (2.9±0.78, p=0.0009) (Fig. C). There was no significant increase in TNFα or IL-6 gene expression or Wet/Dry ratio in the two-gRNA group compared to the diluent group (Fig. D, E) suggesting minimal vector-related inflammation. ChIP-qPCR demonstrated enriched binding of Cas9 to on-target sites (Fig. F, fold enrichment; 17.5±1.7, p=0.02, 18.5±5.8, p=0.02, respectively) compared to the control region whereas the binding to off-target sites were not significantly enriched. <h3>Conclusion</h3> We have achieved targeted in vivo whole organ activation of the IL-10 gene via CRISPR/Cas9-mediated epigenome editing in the lung. This study demonstrates the feasibility of this strategy for optimizing and immunologically enhancing donor organs for transplant.

Cas9 deactivation with photocleavable guide RNAs

Precise control of CRISPR-Cas9 would improve its safety and applicability. Controlled CRISPR inhibition is a promising approach but is complicated by separate inhibitor delivery, incomplete deactivation, and slow kinetics. To overcome these obstacles, we engineered photocleavable guide RNAs (pcRNAs) that endow Cas9 nucleases and base editors with a built-in mechanism for light-based deactivation. pcRNA enabled the fastest (<1min) and most complete (<1% residual indels) approach for Cas9 deactivation. It also exhibited significantly enhanced specificity with wild-type Cas9. Time-resolved deactivation revealed that 12-36h of Cas9 activity or 2-4h of base editor activity was sufficient to achieve high editing efficiency. pcRNA is useful for studies of the cellular response to DNA damage by abolishing sustained cycles of damage and repair that would otherwise desynchronize response trajectories. Together, pcRNA expands the CRISPR toolbox for precision genome editing and studies of DNA damage and repair.

Curiosity-Based Biophysics Projects in a High School Setting with Graduate Student Mentorship

Curiosity-Based Biophysics Projects in a High School Setting with Graduate Student Mentorship

Modulating Cas9 activity for precision gene editing.

The CRISPR/Cas9 is a RNA-guided nuclease complex that can be specifically programmed to target a user-specified DNA sequence. It has been a powerful and effective tool of genome editing. However, off-target activity of the Cas9 nuclease limits its potential use in the correction of inherited diseases and bona fide gene editing. Various protein engineering and guide RNA selection strategies have been utilized to improve Cas9-based genome-editing specificity and efficiency. We, however, have not yet achieved a degree of safety such that Cas9 gene editing approaches could be applicable in clinical settings. Here, we discuss the recently developed and precise gene editing technologies based on spCas9. Furthermore, we describe Cas9 modulating tools to increase the fidelity of the CRISPR/Cas9 system. These studies suggest that there is still a need for pharmaceutical modulation of Cas9 activity during gene editing procedures. Pharmaceutical modulation of Cas9 nuclease activity at on-target or off-target genomic loci could 1 day allow researchers to develop robust and precise therapeutical strategies in gene editing.

In vitro Cleavage and Electrophoretic Mobility Shift Assays for Very Fast CRISPR.

CRISPR-Cas9 has transformed biomedical research and medicine through convenient and targeted manipulation of DNA. Time- and spatially-resolved control over Cas9 activity through the recently developed very fast CRISPR (vfCRISPR) system have facilitated comprehensive studies of DNA damage and repair. Understanding the fundamental principles of Cas9 binding and cleavage behavior is essential before the widespread use of these systems and can be readily accomplished in vitro through both cleavage and electrophoretic mobility shift assays (EMSA). The protocol for in vitro cleavage consists of Cas9 with guide RNA (gRNA) ribonucleoprotein (RNP) formation, followed by incubation with target DNA. For EMSA, this reaction is directly loaded onto an agarose gel for visualization of the target DNA band that is shifted due to binding by the Cas9 RNP. To assay for cleavage, Proteinase K is added to degrade the RNP, allowing target DNA (cleaved and/or uncleaved) to migrate consistently with its molecular weight. Heating at 95°C rapidly inactivates the RNP on demand, allowing time-resolved measurements of Cas9 cleavage kinetics. This protocol facilitates the characterization of the light-activation mechanism of photocaged vfCRISPR gRNA.

Light activation and deactivation of Cas9 for DNA repair studies.

DNA double-strand breaks in DNA (DSBs) are common yet highly detrimental events in living organisms. To repair the damage, each cell requires a coordinated set of DNA damage response (DDR) proteins that can respond quickly, effectively, and precisely. Better understanding of these processes is therefore essential and would require an effective means of inducing targeted DSBs on demand, but previous methods are hampered by limited control over genomic location, timing, or lesion types. Tight spatiotemporal control of CRISPR-Cas9 activity has potential to overcome these limitations, which led to the development of two methods for rapid activation or deactivation of Cas9 using light. In this chapter, we discuss how control of Cas9 can advance DDR studies, describe protocols to control Cas9 activation and deactivation using this new technology, and finally outline three compatible readouts of DNA damage and the cellular response: DSB levels using droplet digital PCR, repair factor localization using ChIP-seq, and insertion-deletion (indel) repair outcomes using Sanger sequencing.

Optical Control of Genome Editing by Photoactivatable Cas9.

The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.

CRISPR-Cas "Non-Target" Sites Inhibit On-Target Cutting Rates.

CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms have been developed to predict gRNA-specific Cas9 activity, a fundamental biological understanding of gRNA-specific activity is lacking. The number of PAM sites in the genome is effectively a large pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose-dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity toward a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates, while increased PAM promiscuity decreases cutting rates. Decreasing the potential search space by increasing PAM specificity provides a path toward improving on-target activity for slower high-fidelity Cas9 variants. Engineering improved PAM specificity to reduce the competitive search space offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept

The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimised CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilising domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes

Prolonged Cas9 activity can hinder genome engineering as it causes off-target effects, genotoxicity, heterogeneous genome-editing outcomes, immunogenicity, and mosaicism in embryonic editing—issues which could be addressed by controlling the longevity of Cas9. Though some temporal controls of Cas9 activity have been developed, only cumbersome systems exist for modifying the lifetime. Here, we have developed a chemogenetic system that brings Cas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of Cas9 by the proteasome. Despite the large size of Cas9, we were able to demonstrate efficient degradation in cells from multiple species. Furthermore, by controlling the Cas9 lifetime, we were able to bias the DNA repair pathways and the genotypic outcome for both templated and nontemplated genome editing. Finally, we were able to dosably control the Cas9 activity and specificity to ameliorate the off-target effects. The ability of this system to change the Cas9 lifetime and, therefore, bias repair pathways and specificity in the desired direction allows precision control of the genome editing outcome.

Development of aptamer-based inhibitors for CRISPR/Cas system.

The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.

A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy

The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.

Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.

Programmable Live‐Cell CRISPR Imaging with Toehold‐Switch‐Mediated Strand Displacement

AbstractThe widespread application of CRISPR‐Cas9 has transformed genome engineering. Nevertheless, the precision to control the targeting activity of Cas9 requires further improvement. We report a toehold‐switch‐based approach to engineer the conformation of single guide RNA (sgRNA) for programmable activation of Cas9. This activation circuit is responsive to multiple inputs and can regulate the conformation of the sgRNA through toehold‐switch‐mediated strand displacement. We demonstrate the orthogonal suppression and activation of Cas9 with orthogonal DNA inputs. Combination of toehold switches leads to a variety of intracellular Cas9 activation programs with simultaneous and orthogonal responses, through which multiple genome loci are displayed in different colors in a controllable manner. This approach provides a new route for programing CRISPR in living cells for genome imaging and engineering.

Programmable Live-Cell CRISPR Imaging with Toehold-Switch-Mediated Strand Displacement.

The widespread application of CRISPR-Cas9 has transformed genome engineering. Nevertheless, the precision to control the targeting activity of Cas9 requires further improvement. We report a toehold-switch-based approach to engineer the conformation of single guide RNA (sgRNA) for programmable activation of Cas9. This activation circuit is responsive to multiple inputs and can regulate the conformation of the sgRNA through toehold-switch-mediated strand displacement. We demonstrate the orthogonal suppression and activation of Cas9 with orthogonal DNA inputs. Combination of toehold switches leads to a variety of intracellular Cas9 activation programs with simultaneous and orthogonal responses, through which multiple genome loci are displayed in different colors in a controllable manner. This approach provides a new route for programing CRISPR in living cells for genome imaging and engineering.

Abstract NG02: Genomic evolution of cancer models: Perils and opportunities

Abstract NG02: Genomic evolution of cancer models: Perils and opportunities

Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On- and Off-Target Damage: Techniques for Detecting, Predicting, and Mitigating the On- and off-target Effects of Cas9 Editing.

Large deletions and genomic re-arrangements are increasingly recognized as common products of double-strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats - CRISPR associated protein 9 (CRISPR/Cas9) on-target sites. Together with well-known off-target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9-editing is necessary to ensure validity of observed phenotypes in Cas9-edited cell-lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide-RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off-targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on- and off-target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.

Decipher the complexity of cis-regulatory regions by a modified Cas9.

Understanding complex mechanisms of human transcriptional regulation remains a major challenge. Classical reporter studies already enabled the discovery of cis-regulatory elements within the non-coding DNA; however, the influence of genomic context and potential interactions are still largely unknown. Using a modified Cas9 activation complex we explore the complexity of renin transcription in its native genomic context. With the help of genomic editing, we stably tagged the native renin on chromosome 1 with the firefly luciferase and stably integrated a programmable modified Cas9 based trans-activation complex (SAM-complex) by lentiviral transduction into human cells. By delivering five specific guide-RNA homologous to specific promoter regions of renin we were able to guide this SAM-complex to these regions of interest. We measured gene expression and generated and compared computational models. SAM complexes induced activation of renin in our cells after renin specific guide-RNA had been provided. All possible combinations of the five guides were subjected to model analysis in linear models. Quantifying the prediction error and the calculation of an estimator of the relative quality of the statistical models for our given set of data revealed that a model incorporating interactions in the proximal promoter is the superior model for explanation of the data. By applying our combined experimental and modelling approach we can show that interactions occur within the selected sequences of the proximal renin promoter region. This combined approach might potentially be useful to investigate other genomic regions. Our findings may help to better understand the transcriptional regulation of human renin.

The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly invitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.