scholarly journals A rapid and tunable method to temporally control Cas9 expression enables the identification of essential genes and the interrogation of functional gene interactions in vitro and in vivo.

2015 ◽  
Author(s):  
Serif Senturk ◽  
Nitin H Shirole ◽  
Dawid D. Nowak ◽  
Vincenzo Corbo ◽  
Alexander Vaughan ◽  
...  
Keyword(s):  
Gene Editing ◽  
Essential Genes ◽  
Gene Interactions ◽  
Guide Rna ◽  
Functional Interactions ◽  
Cell Specificity ◽  
Efficient Gene ◽  
Systematic Identification

The Cas9/CRISPR system is a powerful tool for studying gene function. Here we describe a method that allows temporal control of Cas9/CRISPER activity based on conditional CAS9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 enables conditional rapid and reversible Cas9 expression in vitro and efficient gene-editing in the presence of a guide RNA. Further, we show that this strategy can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest, without the latter being co-modulated. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic identification of essential genes and the interrogation of genes functional interactions.

Advances in therapeutic application of CRISPR-Cas9

2019 ◽  
Vol 19 (3) ◽  
pp. 164-174 ◽  
Author(s):  
Jinyu Sun ◽  
Jianchu Wang ◽  
Donghui Zheng ◽  
Xiaorong Hu
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Malignant Tumor ◽  
Gene Editing ◽  
Genome Engineering ◽  
Therapeutic Application ◽  
Adaptive Immune ◽  
Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

scholarly journals Polyvalent Guide RNAs for CRISPR Antivirals

2021 ◽  
Author(s):  
Rammyani Bagchi ◽  
Rachel Tinker-Kulberg ◽  
Tinku Supakar ◽  
Sydney Chamberlain ◽  
Ayalew Ligaba-Osena ◽  
...  
Keyword(s):  
Gene Editing ◽  
Higher Plants ◽  
Sequence Divergence ◽  
Detection Sensitivity ◽  
Clinical Strain ◽  
Viral Detection ◽  
Guide Rna ◽  
Target Sites

ABSTRACTCRISPR biotechnologies, where CRISPR effectors recognize and degrade specific nucleic acid targets that are complementary to their guide RNA (gRNA) cofactors, have been primarily used as a tool for precision gene editing1 but possess an emerging potential for novel antiviral diagnostics, prophylactics, and therapeutics.2–5 In gene editing applications, significant efforts are made to limit the natural tolerance of CRISPR effectors for nucleic acids with imperfect complementarity to their gRNAs in order to prevent degradation and mutation at unintended or “off-target” sites; here we exploit those tolerances to engineer gRNAs that are optimized to promote activity at multiple viral target sites, simultaneously, given that multiplexed targeting is a critical tactic for improving viral detection sensitivity,3 expanding recognition of clinical strain variants,6 and suppressing viral mutagenic escape from CRISPR antivirals.7 We demonstrate in vitro and in higher plants that single “polyvalent” gRNAs (pgRNAs) in complex with CRISPR effectors Cas9 or Cas13 can effectively degrade pairs of viral targets with significant sequence divergence (up to 40% nucleotide differences) that are prevalent in viral genomes. We find that CRISPR antivirals using pgRNAs can robustly suppress the propagation of plant RNA viruses, in vivo, better than those with a “monovalent” gRNA counterpart. These results represent a powerful new approach to gRNA design for antiviral applications that can be readily incorporated into current viral detection and therapeutic strategies, and highlight the need for specific approaches and tools that can address the differential requirements of precision gene editing vs. CRISPR antiviral applications in order to mature these promising biotechnologies.

scholarly journals Near-infrared upconversion–activated CRISPR-Cas9 system: A remote-controlled gene editing platform

2019 ◽  
Vol 5 (4) ◽  
pp. eaav7199 ◽  
Author(s):  
Yongchun Pan ◽  
Jingjing Yang ◽  
Xiaowei Luan ◽  
Xinli Liu ◽  
Xueqing Li ◽  
...  
Keyword(s):  
Gene Editing ◽  
Near Infrared ◽  
Cancer Therapeutics ◽  
Guide Rna ◽  
System A ◽  
Nir Light ◽  
Targeted Gene Editing ◽  
Daunting Challenge

As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light–responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as “nanotransducers” that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9. In addition, by preparing a single guide RNA targeting a tumor gene (polo-like kinase-1), our strategies have successfully inhibited the proliferation of tumor cell via NIR light–activated gene editing both in vitro and in vivo. Overall, this exogenously controlled method presents enormous potential for targeted gene editing in deep tissues and treatment of a myriad of diseases.

scholarly journals A systematic evaluation of data processing and problem formulation of CRISPR off-target site prediction

2021 ◽  
Author(s):  
Ofir Yaish ◽  
Maor Asif ◽  
Yaron Orenstein
Keyword(s):  
Data Processing ◽  
Gene Editing ◽  
Target Site ◽  
Systematic Evaluation ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Sites ◽  
Evaluation Of Data

AbstractCRISPR/Cas9 system is widely used in a broad range of gene-editing applications. While this gene-editing technique is quite accurate in the target region, there may be many unplanned off-target edited sites. Consequently, a plethora of computational methods have been developed to predict off-target cleavage sites given a guide RNA and a reference genome. However, these methods are based on small-scale datasets (only tens to hundreds of off-target sites) produced by experimental techniques to detect off-target sites with a low signal-to-noise ratio. Recently, CHANGE-seq, a new in vitro experimental technique to detect off-target sites, was used to produce a dataset of unprecedented scale and quality (more than 200,000 off-target sites over 110 guide RNAs). In addition, the same study included GUIDE-seq experiments for 58 of the guide RNAs to produce in vivo measurements of off-target sites. Here, we fill the gap in previous computational methods by utilizing these data to perform a systematic evaluation of data processing and formulation of the CRISPR off-target site prediction problem. Our evaluations show that data transformation as a pre-processing phase is critical prior to model training. Moreover, we demonstrate the improvement gained by adding potential inactive off-target sites to the training datasets. Furthermore, our results point to the importance of adding the number of mismatches between the guide RNA and the off-target site as a feature. Finally, we present predictive off-target in vivo models based on transfer learning from in vitro. Our conclusions will be instrumental to any future development of an off-target predictor based on high-throughput datasets.

scholarly journals Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing

2020 ◽  
Author(s):  
Fan Li ◽  
Kristof Wing ◽  
Jiang-Hui Wang ◽  
Chi D. Luu ◽  
James A. Bender ◽  
...  
Keyword(s):  
Gene Editing ◽  
Vector System ◽  
Gene Correction ◽  
Retinal Cells ◽  
Dual Vector ◽  
Vector Systems ◽  
Efficient Gene ◽  
Significant Difference

ABSTRACTCRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and sgRNA targeting YFP and a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre::Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vitro and in vivo.

scholarly journals CRISPR Gene-Editing Models Geared Toward Therapy for Hereditary and Developmental Neurological Disorders

2021 ◽  
Vol 9 ◽  
Author(s):  
Poh Kuan Wong ◽  
Fook Choe Cheah ◽  
Saiful Effendi Syafruddin ◽  
M. Aiman Mohtar ◽  
Norazrina Azmi ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Neurological Disorders ◽  
Gene Editing ◽  
Fragile X ◽  
Genetic Diseases ◽  
Basic Principles ◽  
Short Palindromic Repeat

Hereditary or developmental neurological disorders (HNDs or DNDs) affect the quality of life and contribute to the high mortality rates among neonates. Most HNDs are incurable, and the search for new and effective treatments is hampered by challenges peculiar to the human brain, which is guarded by the near-impervious blood-brain barrier. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), a gene-editing tool repurposed from bacterial defense systems against viruses, has been touted by some as a panacea for genetic diseases. CRISPR has expedited the research into HNDs, enabling the generation of in vitro and in vivo models to simulate the changes in human physiology caused by genetic variation. In this review, we describe the basic principles and workings of CRISPR and the modifications that have been made to broaden its applications. Then, we review important CRISPR-based studies that have opened new doors to the treatment of HNDs such as fragile X syndrome and Down syndrome. We also discuss how CRISPR can be used to generate research models to examine the effects of genetic variation and caffeine therapy on the developing brain. Several drawbacks of CRISPR may preclude its use at the clinics, particularly the vulnerability of neuronal cells to the adverse effect of gene editing, and the inefficiency of CRISPR delivery into the brain. In concluding the review, we offer some suggestions for enhancing the gene-editing efficacy of CRISPR and how it may be morphed into safe and effective therapy for HNDs and other brain disorders.

scholarly journals Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mark Austin Hanson ◽  
Anna Dostálová ◽  
Camilla Ceroni ◽  
Mickael Poidevin ◽  
Shu Kondo ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Fungal Pathogens ◽  
Gene Editing ◽  
Cationic Peptides ◽  
Gram Negative Bacteria ◽  
Biological Processes ◽  
Gram Negative ◽  
Bacteria And Fungi

Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading microorganisms. These short, cationic peptides have been implicated in many biological processes, primarily involving innate immunity. In vitro studies have shown AMPs kill bacteria and fungi at physiological concentrations, but little validation has been done in vivo. We utilized CRISPR gene editing to delete most known immune-inducible AMPs of Drosophila, namely: 4 Attacins, 2 Diptericins, Drosocin, Drosomycin, Metchnikowin and Defensin. Using individual and multiple knockouts, including flies lacking these ten AMP genes, we characterize the in vivo function of individual and groups of AMPs against diverse bacterial and fungal pathogens. We found that Drosophila AMPs act primarily against Gram-negative bacteria and fungi, contributing either additively or synergistically. We also describe remarkable specificity wherein certain AMPs contribute the bulk of microbicidal activity against specific pathogens, providing functional demonstrations of highly specific AMP-pathogen interactions in an in vivo setting.

scholarly journals Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

2018 ◽  
Vol 115 (19) ◽  
pp. 4903-4908 ◽  
Author(s):  
Hong-Xia Wang ◽  
Ziyuan Song ◽  
Yeh-Hsing Lao ◽  
Xin Xu ◽  
Jing Gong ◽  
...  
Keyword(s):  
Gene Editing ◽  
Transfection Efficiency ◽  
Gene Activation ◽  
Cell Types ◽  
Biological Research ◽  
Knock Out ◽  
Safe Delivery ◽  
Pegylated Nanoparticles

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

piCRISPR: Physically Informed Features Improve Deep Learning Models for CRISPR/Cas9 Off-Target Cleavage Prediction

2021 ◽  
Author(s):  
Florian Störtz ◽  
Jeffrey Mak ◽  
Peter Minary
Keyword(s):  
Deep Learning ◽  
Gene Editing ◽  
Target Prediction ◽  
Model Space ◽  
Chromatin Accessibility ◽  
Sequence Context ◽  
Prediction Algorithms ◽  
Cleavage Assay

CRISPR/Cas programmable nuclease systems have become ubiquitous in the field of gene editing. With progressing development, applications in in vivo therapeutic gene editing are increasingly within reach, yet limited by possible adverse side effects from unwanted edits. Recent years have thus seen continuous development of off-target prediction algorithms trained on in vitro cleavage assay data gained from immortalised cell lines. Here, we implement novel deep learning algorithms and feature encodings for off-target prediction and systematically sample the resulting model space in order to find optimal models and inform future modelling efforts. We lay emphasis on physically informed features, hence terming our approach piCRISPR, which we gain on the large, diverse crisprSQL off-target cleavage dataset. We find that our best-performing model highlights the importance of sequence context and chromatin accessibility for cleavage prediction and outperforms state-of-the-art prediction algorithms in terms of area under precision-recall curve.

scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

Sign in / Sign up

Export Citation Format

Share Document