CRISPR-based Technologies Research Articles

Genetic Knockout of TE Insertions by CRISPR-Cas9.

Transposable elements (TEs) are abundant in the genome, and specific insertions may be co-opted to act as coding or noncoding functional elements. CRISPR-based genome editing technologies enable functional studies of TE insertions in cell lines. Here, we describe the use of CRISPR-Cas9 to create and validate genetic knockouts of TEs in mammalian cell lines.

In vivo genome editing using novel AAV-PHP variants rescues motor function deficits and extends survival in a SOD1-ALS mouse model.

CRISPR-based gene editing technology represents a promising approach to deliver therapies for inherited disorders, including amyotrophic lateral sclerosis (ALS). Toxic gain-of-function superoxide dismutase 1 (SOD1) mutations are responsible for ~20% of familial ALS cases. Thus, current clinical strategies to treat SOD1-ALS are designed to lower SOD1 levels. Here, we utilized AAV-PHP.B variants to deliver CRISPR-Cas9 guide RNAs designed to disrupt the human SOD1 (huSOD1) transgene in SOD1G93A mice. A one-time intracerebroventricular injection of AAV.PHP.B-huSOD1-sgRNA into neonatal H11Cas9 SOD1G93A mice caused robust and sustained mutant huSOD1 protein reduction in the cortex and spinal cord, and restored motor function. Neonatal treatment also reduced spinal motor neuron loss, denervation at neuromuscular junction (NMJ) and muscle atrophy, diminished axonal damage and preserved compound muscle action potential throughout the lifespan of treated mice. SOD1G93A treated mice achieved significant disease-free survival, extending lifespan by more than 110 days. Importantly, a one-time intrathecal or intravenous injection of AAV.PHP.eB-huSOD1-sgRNA in adult H11Cas9 SOD1G93A mice, immediately before symptom onset, also extended lifespan by at least 170 days. We observed substantial protection against disease progression, demonstrating the utility of our CRISPR editing preclinical approach for target evaluation. Our approach uncovered key parameters (e.g., AAV capsid, Cas9 expression) that resulted in improved efficacy compared to similar approaches and can also serve to accelerate drug target validation.

CRISPR Activation/Interference Screen to Identify Genetic Networks in HDAC-Inhibitor-Resistant Cells.

Epigenetic alterations have been identified in various tumor types. In part, these alterations are mediated via increased histone deacetylase activity. Although preclinical results of monotherapies with histone deacetylase inhibitors (HDACi) are promising, success in clinical trials is limited. Reasons for these limitations may be de novo or acquired resistance to HDAC inhibitors that could be overcome with rational combination therapies. This requires knowledge of resistance mechanism along with the involved genetic networks. One way to identify such genetic networks is the implementation of a CRISPR-based technology allowing transcriptional repression (CRISPRi) and activation (CRISPRa) at a genome-wide scale. We describe a simple approach to amplify and validate sgRNA libraries, generate a myeloid progenitor cell line expressing catalytically dead Cas9 (dCas9) fusion proteins with transcriptional effectors to repress or activate genetic regions of interest and demonstrate a complementary genome-wide HDACi resistance screening approach. Furthermore, we present bioinformatics tools for quality control and analysis of the sequencing data.

684 Correction of the cystic fibrosis transmembrane conductance regulator 1717-1G >A splicing mutation through CRISPR-based technology

684 Correction of the cystic fibrosis transmembrane conductance regulator 1717-1G >A splicing mutation through CRISPR-based technology

Genome Editing Approaches with CRISPR/Cas9 for Cancer Treatment: Critical Appraisal of Preclinical and Clinical Utility, Challenges, and Future Research.

The increasing burden on human malignant diseases became a major concern for healthcare practitioners, that must deal with tumor relapse and the inability to efficiently treat metastasis, in addition to side effects. Throughout the decades, many therapeutic strategies have been employed to improve the clinical outcomes of cancer patients and great efforts have been made to develop more efficient and targeted medicines. The malignant cell is characterized by genetic and epigenetic modifications, therefore targeting those specific drivers of carcinogenesis is highly desirable. Among the genome editing technologies, CRISPR/Cas9 stood as a promising candidate for cancer treatment alternatives, due to its low complexity design. First described as a defense mechanism of bacteria against invading foreign DNA, later it was shown that CRISPR components can be engineered to target specific DNA sequences in a test tube, a discovery that was awarded later with the Nobel Prize in chemistry for its rapid expansion as a reliable genome editing tool in many fields of research, including medicine. The present paper aims of describing CRISPR/Cas9 potential targets for malignant disorders, and the approaches used for achieving this goal. Aside from preclinical studies, we also present the clinical trials that use CRISPR-based technology for therapeutic purposes of cancer. Finally, a summary of the presented studies adds a more focused view of the therapeutic value CRISPR/Cas9 holds and the associated shortcomings.

CRISPR-Based Genome Editing and Its Applications in Woody Plants.

CRISPR/Cas-based genome editing technology provides straightforward, proficient, and multifunctional ways for the site-directed modification of organism genomes and genes. The application of CRISPR-based technology in plants has a vast potential value in gene function research, germplasm innovation, and genetic improvement. The complexity of woody plants genome may pose significant challenges in the application and expansion of various new editing techniques, such as Cas9, 12, 13, and 14 effectors, base editing, particularly for timberland species with a long life span, huge genome, and ploidy. Therefore, many novel optimisms have been drawn to molecular breeding research based on woody plants. This review summarizes the recent development of CRISPR/Cas applications for essential traits, including wood properties, flowering, biological stress, abiotic stress, growth, and development in woody plants. We outlined the current problems and future development trends of this technology in germplasm and the improvement of products in woody plants.

Towards the diagnosis of dengue virus and its serotypes using designed CRISPR/Cas13 gRNAs.

Dengue Virus (DENV) is a mosquito-borne virus that is prevalent in the world's tropical and subtropical regions. Therefore, early detection and surveillance can help in the management of this disease. Current diagnostic methods rely primarily on ELISA, PCR, and RT-PCR, among others, which can only be performed in specialized laboratories and require sophisticated instruments and technical expertise. CRISPR-based technologies on the other hand have field-deployable viral diagnostics capabilities that could be used in the development of point-of-care molecular diagnostics. The first step in the field of CRISPR-based virus diagnosis is to design and screen gRNAs for high efficiency and specificity. In the present study, we employed a bioinformatics approach to design and screen DENV CRISPR/Cas13 gRNAs for conserved and serotype-specific variable genomic regions in the DENV genome. We identified one gRNA sequence specific for each of the lncRNA and NS5 regions and identified one gRNA against each of DENV1, DENV2, DENV3, and DENV4 to distinguish the four DENV serotypes. These CRISPR/Cas13 gRNA sequences will be useful in diagnosing the dengue virus and its serotypes for in vitro validation and diagnostics.

Genomic and epigenetic landscapes drive CRISPR-based genome editing in Bifidobacterium

Bifidobacterium is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in B. animalis subsp. lactis, demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion in tetW to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple B. lactis strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among B. lactis strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to Bifidobacterium longum subsp. infantis to emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics.

Abstract 1644: Generation and validation of humanized GARP mice for testing novel anti-human GARP antibodies

Abstract GARP is a type I transmembrane protein expressed on regulatory T cells (Tregs) and platelets. GARP expression has been shown to play an important role in the modulation of latent TGF-β1 activity by regulatory T lymphocytes (Tregs), and thus, GARP has emerged as an important regulator of peripheral immune tolerance. While GARP expression is critical for homeostasis, increased GARP expression on Tregs is associated with elevated immunosuppression in the context of tumor biology. Because of these immunomodulatory roles, targeting GARP in the context of oncologic or other diseases is of significant interest. Thus, the development of mouse models for safety and efficacy evaluation of GARP-targeted therapeutics prior to clinical trials is needed. To facilitate assessment of novel anti-human GARP antibodies, Biocytogen has generated a humanized GARP mouse model for in vitro functional validation and in vivo efficacy evaluation assays. Using CRISPR-based gene editing technology, the exons of the mouse Garp gene that encode the extracellular domain were replaced by the human GARP counterpart. mRNA Expression of the human GARP extracellular domain was confirmed in splenocytes isolated from homozygous humanized mice. Expression of human GARP was readily detected in FoxP3+ Treg cells isolated from the spleen of humanized GARP mice as assessed by flow cytometry. Importantly, the frequency of FoxP3+ Tregs and other mature leukocyte subpopulations, including T and B lymphocytes, Natural killer cells, dendritic cells, granulocytes, and monocytes/macrophages, was also similar in the blood and spleen of humanized GARP mice compared to wild-type mice. Subsequent in vivo efficacy assays using the MC38 tumor model demonstrated efficacy of the anti-human GARP antibody in slowing tumor growth in humanized GARP mice. Furthermore, combination of anti-human GARP and anti-mouse PD-1/PD-L1 blockade resulted in a significantly greater tumor inhibition than monotherapies. In summary, GARP humanized mice are a powerful platform for preclinical evaluation of anti-human GARP antibodies for cancer treatment. Citation Format: Ruili Lyu, Huilin Li, Jenna Frame, Yuelei Shen. Generation and validation of humanized GARP mice for testing novel anti-human GARP antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1644.

CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells.

The development of CRISPR-based gene-editing technologies has brought an unprecedented revolution in the field of genome engineering. Cas12a, a member of the Class 2 Type V CRISPR-associated endonuclease family distinct from Cas9, has been repurposed and developed into versatile gene-editing tools with distinct PAM recognition sites and multiplexed gene targeting capability. However, with current CRISPR/Cas12a technologies, it remains a challenge to perform efficient and precise genome editing of long sequences in mammalian cells. To address this limitation, we utilized phage recombination enzymes and developed an efficient CRISPR/Cas12a tool for multiplexed precision editing in mammalian cells. Through protein engineering, we were able to recruit phage recombination proteins to Cas12a to enhance its homology-directed repair efficiencies. Our phage-recombination-assisted Cas12a system achieved up to 3-fold improvements for kilobase-scale knock-ins in human cells without compromising the specificity of the enzyme. The performance of this system compares favorably against Cas9 references, the commonly used enzyme for gene-editing tasks, with improved specificity. Additionally, we demonstrated multi-target editing with similar improved activities thanks to the RNA-processing activity of the Cas12a system. This compact, multi-target editing tool has the potential to assist in understanding multi-gene interactions. In particular, it paves the way for a gene therapy method for human diseases that complements existing tools and is suitable for polygenic disorders and diseases requiring long-sequence corrections.

Dissecting Plant Gene Functions Using CRISPR Toolsets for Crop Improvement.

The CRISPR-based gene editing technology has become more and more powerful in genome manipulation for agricultural breeding, with numerous improved toolsets springing up. In recent years, many CRISPR toolsets for gene editing, such as base editors (BEs), CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and plant epigenetic editors (PEEs), have been developed to clarify gene function and full-level gene regulation. Here, we comprehensively summarize the application and capacity of the different CRISPR toolsets in the study of plant gene expression regulation, highlighting their potential application in gene regulatory networks' analysis. The general problems in CRISPR application and the optimal solutions in the existing schemes for high-throughput gene function analysis are also discussed. The CRISPR toolsets targeting gene manipulation discussed here provide new solutions for further genetic improvement and molecular breeding of crops.

Generation of a recessive dystrophic epidermolysis bullosa mouse model with patient-derived compound heterozygous mutations

AbstractRecessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic disease of the skin caused by mutations in the COL7A1 gene. The majority of patients with RDEB harbor compound heterozygous mutations—two distinct mutations on each chromosome—without any apparent hotspots in the COL7A1 mutation pattern. This situation has made it challenging to establish a reliable RDEB mouse model with mutations that accurately mimic the genomic background of patients. Here, we established an RDEB mouse model harboring patient-type mutations in a compound heterozygous manner, using the CRISPR-based genome-editing technology i-GONAD. We selected two mutations, c.5818delC and E2857X, that have frequently been identified in cohorts of Japanese patients with RDEB. These mutations were introduced into the mouse genome at locations corresponding to those identified in patients. Mice homozygous for the 5818delC mutation developed severe RDEB-like phenotypes and died immediately after birth, whereas E2857X homozygous mice did not have a shortened lifespan compared to wild-type mice. Adult E2857X homozygous mice showed hair abnormalities, syndactyly, and nail dystrophy; these findings indicate that E2857X is indeed pathogenic in mice. Mice with the c.5818delC/E2857X compound heterozygous mutation presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. Single-cell RNA sequencing further clarified that the intrafollicular keratinocytes in c.5818delC/E2857X compound heterozygous mice exhibited abnormalities in cell cycle regulation. The proposed strategy to produce compound heterozygous mice, in addition to the established mouse line, will facilitate research on RDEB pathogenesis to develop a cure for this devastating disease.

Molecular and Computational Strategies to Increase the Efficiency of CRISPR-Based Techniques.

The prokaryote-derived Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas mediated gene editing tools have revolutionized our ability to precisely manipulate specific genome sequences in plants and animals. The simplicity, precision, affordability, and robustness of this technology have allowed a myriad of genomes from a diverse group of plant species to be successfully edited. Even though CRISPR/Cas, base editing, and prime editing technologies have been rapidly adopted and implemented in plants, their editing efficiency rate and specificity varies greatly. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9-derived technologies and their implications on enhancing editing efficiency. We highlight the major efforts of engineering Cas9, Cas12a, Cas12b, and Cas12f proteins aiming to improve their efficiencies. We also provide a perspective on the global future of agriculturally based products using DNA-free CRISPR/Cas techniques. The improvement of CRISPR-based technologies efficiency will enable the implementation of genome editing tools in a variety of crop plants, as well as accelerate progress in basic research and molecular breeding.

Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations.

Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into these mutations’ molecular properties and implications in tumorigenesis. The generation of such models harboring the alterations is feasible with the CRISPR-based gene editing technology. Moreover, the development of new CRISPR applications, particularly DNA base and prime editing, has considerably improved the precision and versatility of gene editing. Here, we employed the prime editing tool to revert a TP53 missense C > T mutation (L194F) in a T47D luminal A breast cancer cell line. In parallel, this prime editing tool was also utilized to introduce the L194F mutation in HEK293T cells. To assess the prime editing efficiency in both cell lines, we first performed Sanger sequencing in the prime-edited cells pool and single cell-derived clones. However, the Sanger sequencing approach did not detect any base substitution in these cell lines. Next, by employing the more sensitive amplicon target sequencing, we managed to identify the expected substitution in these T47D and HEK293T cells, albeit the editing efficiency was low. In light of these findings, we discussed the technical aspects and provided suggestions for improve the prime editing workflow and efficiency for future experiments.

Single cell lineage reconstruction using distance-based algorithms and the R package, DCLEAR

BackgroundDCLEAR is an R package used for single cell lineage reconstruction. The advances of CRISPR-based gene editing technologies have enabled the prediction of cell lineage trees based on observed edited barcodes from each cell. However, the performance of existing reconstruction methods of cell lineage trees was not accessed until recently. In response to this problem, the Allen Institute hosted the Cell Lineage Reconstruction Dream Challenge in 2020 to crowdsource relevant knowledge from across the world. Our team won sub-challenges 2 and 3 in the challenge competition.ResultsThe DCLEAR package contained the R codes, which was submitted in response to sub-challenges 2 and 3. Our method consists of two steps: (1) distance matrix estimation and (2) the tree reconstruction from the distance matrix. We proposed two novel methods for distance matrix estimation as outlined in the DCLEAR package. Using our method, we find that two of the more sophisticated distance methods display a substantially improved level of performance compared to the traditional Hamming distance method. DCLEAR is open source and freely available from R CRAN and from under the GNU General Public License, version 3.ConclusionsDCLEAR is a powerful resource for single cell lineage reconstruction.

In vivo prime editing of a metabolic liver disease in mice.

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain (PE2ΔRnH) and an intein-split construct (PE2 p.1153) for adeno-associated virus-mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2ΔRnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pahenu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 1014 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.

Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing

Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called ‘BALL’, enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.

Vaccines for Human Schistosomiasis: Recent Progress, New Developments and Future Prospects.

Schistosomiasis, caused by human trematode blood flukes (schistosomes), remains one of the most prevalent and serious of the neglected tropical parasitic diseases. Currently, treatment of schistosomiasis relies solely on a single drug, the anthelmintic praziquantel, and with increased usage in mass drug administration control programs for the disease, the specter of drug resistance developing is a constant threat. Vaccination is recognized as one of the most sustainable options for the control of any pathogen, but despite the discovery and reporting of numerous potentially promising schistosome vaccine antigens, to date, no schistosomiasis vaccine for human or animal deployment is available. This is despite the fact that Science ranked such an intervention as one of the top 10 vaccines that need to be urgently developed to improve public health globally. This review summarizes current progress of schistosomiasis vaccines under clinical development and advocates the urgent need for the establishment of a revolutionary and effective anti-schistosome vaccine pipeline utilizing cutting-edge technologies (including developing mRNA vaccines and exploiting CRISPR-based technologies) to provide novel insight into future vaccine discovery, design, manufacture and deployment.

The Trend of CRISPR-Based Technologies in COVID-19 Disease: Beyond Genome Editing.

Biotechnological approaches have always sought to utilize novel and efficient methods in the prevention, diagnosis, and treatment of diseases. This science has consistently tried to revolutionize medical science by employing state-of-the-art technologies in genomic and proteomic engineering. CRISPR–Cas system is one of the emerging techniques in the field of biotechnology. To date, the CRISPR–Cas system has been extensively applied in gene editing, targeting genomic sequences for diagnosis, treatment of diseases through genomic manipulation, and in creating animal models for preclinical researches. With the emergence of the COVID-19 pandemic in 2019, there is need for the development and modification of novel tools such as the CRISPR–Cas system for use in diagnostic emergencies. This system can compete with other existing biotechnological methods in accuracy, precision, and wide performance that could guarantee its future in these conditions. In this article, we review the various platforms of the CRISPR–Cas system meant for SARS-CoV-2 diagnosis, anti-viral therapeutic procedures, producing animal models for preclinical studies, and genome-wide screening studies toward drug and vaccine development.

From Genome Sequencing to CRISPR-Based Genome Editing for Climate-Resilient Forest Trees.

Due to the economic and ecological importance of forest trees, modern breeding and genetic manipulation of forest trees have become increasingly prevalent. The CRISPR-based technology provides a versatile, powerful, and widely accepted tool for analyzing gene function and precise genetic modification in virtually any species but remains largely unexplored in forest species. Rapidly accumulating genetic and genomic resources for forest trees enabled the identification of numerous genes and biological processes that are associated with important traits such as wood quality, drought, or pest resistance, facilitating the selection of suitable gene editing targets. Here, we introduce and discuss the latest progress, opportunities, and challenges of genome sequencing and editing for improving forest sustainability.