scholarly journals Quantifying CRISPR off-target effects

2019 ◽  
Vol 3 (3) ◽  
pp. 327-334 ◽  
Author(s):  
Soragia Athina Gkazi
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Severe Combined Immunodeficiency ◽  
Gene Therapies ◽  
Genome Wide ◽  
Large Indels ◽  
Target Effects

Abstract Recent advances in the era of genetic engineering have significantly improved our ability to make precise changes in the genomes of human cells. Throughout the years, clinical trials based on gene therapies have led to the cure of diseases such as X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID) and Wiskott–Aldrich syndrome. Despite the success gene therapy has had, there is still the risk of genotoxicity due to the potential oncogenesis introduced by utilising viral vectors. Research has focused on alternative strategies like genome editing without viral vectors as a means to reduce genotoxicity introduced by the viral vectors. Although there is an extensive use of RNA-guided genome editing via the clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein-9 (Cas9) technology for biomedical research, its genome-wide target specificity and its genotoxic side effects remain controversial. There have been reports of on- and off-target effects created by CRISPR–Cas9 that can include small and large indels and inversions, highlighting the potential risk of insertional mutagenesis. In the last few years, a plethora of in silico, in vitro and in vivo genome-wide assays have been introduced with the sole purpose of profiling these effects. Here, we are going to discuss the genotoxic obstacles in gene therapies and give an up-to-date overview of methodologies for quantifying CRISPR–Cas9 effects.

scholarly journals In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

2018 ◽  
Author(s):  
Pinar Akcakaya ◽  
Maggie L. Bobbin ◽  
Jimmy A. Guo ◽  
Jose M. Lopez ◽  
M. Kendell Clement ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Strong Support ◽  
Guide Rna ◽  
Genome Wide ◽  
Highly Sensitive ◽  
Further Development ◽  
Target Effects ◽  
Cas Gene

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

scholarly journals A Mixed-Surface Polyamidoamine Dendrimer for In Vitro and In Vivo Delivery of Large Plasmids

Pharmaceutics ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 619
Author(s):  
Bhairavi Srinageshwar ◽  
Maria Florendo ◽  
Brittany Clark ◽  
Kayla Johnson ◽  
Nikolas Munro ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Viral Vector ◽  
Direct Injection ◽  
Gene Therapies ◽  
Large Plasmids ◽  
Surface Modified ◽  
The Brain ◽  
In Vivo Delivery

Drug delivery to the brain is highly hindered by the presence of the blood–brain barrier (BBB), which prevents the entry of many potential drugs/biomolecules into the brain. One of the current strategies to achieve gene therapy for neurodegenerative diseases involves direct injection of a viral vector into the brain. There are various disadvantages of viral vectors, including limitations of cargo size and safety concerns. Nanomolecules, such as dendrimers, serve as an excellent alternative to viral delivery. In this study, as proof-of-concept, we used a surface-modified dendrimer complex and delivered large plasmids to cells in vitro and in vivo in healthy rats via intracranial injection. The dendrimers were biodegradable by chemicals found within cells and toxicity assays revealed that the modified dendrimers were much less toxic than unmodified amine-surface dendrimers. As mentioned in our previous publication, these dendrimers with appropriately modified surfaces are safe, can deliver large plasmids to the brain, and can overcome the cargo size limitations associated with viral vectors. The biocompatibility of this dendritic nanomolecule and the ability to finely tune its surface chemistry provides a gene delivery system that could facilitate future in vivo cellular reprograming and other gene therapies.

scholarly journals Engineering an RNA-based tissue-specific platform for genetic editing through use of a miRNA-enabled Cas12a

2020 ◽  
Author(s):  
Rasmus Møller ◽  
Kohei Oishi ◽  
Benjamin R. tenOever
Keyword(s):  
Viral Vectors ◽  
High Efficiency ◽  
Specific Activity ◽  
Rna Virus ◽  
Immunological Response ◽  
Immunological Responses ◽  
A Cell ◽  
Target Effects

ABSTRACTThe capacity to edit genomes using CRISPR-Cas systems holds immense potential for countless genetic-based diseases. However, one significant impediment preventing broad therapeutic utilization is in vivo delivery. While genetic editing at a single cell level in vitro can be achieved with high efficiency, the capacity to utilize these same biologic tools in a desired tissue in vivo remains challenging. Non-integrating RNA virus-based vectors constitute efficient platforms for transgene expression and surpass several barriers to in vivo delivery. However, the broad tissue tropism of viral vectors raises the concern for off-target effects. Moreover, prolonged expression of the Cas proteins, regardless of delivery method, can accumulate aberrant RNAs leading to unwanted immunological responses. In an effort to circumvent these shortcomings, here we describe a versatile RNA virus-based technology that can achieve cell-specific activity and self-inactivation by combining host microRNA (miRNA) biology with the CRISPR-Cas12a RNA-guided nuclease. Exploiting the RNase activity of Cas12a, we generated a vector that self-inactivates upon delivery of Cas12a and an accompanying CRISPR RNA (crRNA). Furthermore, we show that maturation of the crRNA can be made dependent on cell-specific miRNAs, which confers cell-specificity. We demonstrate that this genetic editing circuit delivers diminished yet sufficient levels of Cas12a to achieve effective genome editing whilst inducing a minimal immunological response. It can also function in a cell-specific manner thereby facilitating in vivo editing and mitigating the risk of unwanted, off-target effects.

scholarly journals Recent Advances in CRISPR/Cas9 Delivery Strategies

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 839 ◽  
Author(s):  
Bon Ham Yip
Keyword(s):  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Gene Editing ◽  
Ex Vivo ◽  
Extracellular Vesicle ◽  
Delivery Methods ◽  
Delivery Strategies ◽  
In Vivo Delivery

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the field of gene editing. Continuous efforts in developing this technology have enabled efficient in vitro, ex vivo, and in vivo gene editing through a variety of delivery strategies. Viral vectors are commonly used in in vitro, ex vivo, and in vivo delivery systems, but they can cause insertional mutagenesis, have limited cloning capacity, and/or elicit immunologic responses. Physical delivery methods are largely restricted to in vitro and ex vivo systems, whereas chemical delivery methods require extensive optimization to improve their efficiency for in vivo gene editing. Achieving a safe and efficient in vivo delivery system for CRISPR/Cas9 remains the most challenging aspect of gene editing. Recently, extracellular vesicle-based systems were reported in various studies to deliver Cas9 in vitro and in vivo. In comparison with other methods, extracellular vesicles offer a safe, transient, and cost-effective yet efficient platform for delivery, indicating their potential for Cas9 delivery in clinical trials. In this review, we first discuss the pros and cons of different Cas9 delivery strategies. We then specifically review the development of extracellular vesicle-mediated gene editing and highlight the strengths and weaknesses of this technology.

A ComParison of Retroviral Integration Sites in Donor T-Lymphocytes In Vivo and In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1403-1403
Author(s):  
Frank A. Giordano ◽  
Stephanie Laufs ◽  
Boris Fehse ◽  
K. Zsuzsanna Nagy ◽  
Agnes Hotz-Wagenblatt ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Insertional Mutagenesis ◽  
Homo Sapiens ◽  
Severe Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Retroviral Integration ◽  
Gene Therapy Trial ◽  
Integration Sites

Abstract Genetically modified T-Lymphocytes (TLCs) have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused TLCs have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Yet, reports on insertional mutagenesis in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. We investigated retroviral integration sites in vitro and in vivo. Therefore, donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN for a clinical HSV-Tk study were examined. TLCs of four different donors as well as whole blood samples of two patients transplanted with donor TLCs were analyzed either using highly sensitive and specific ligation-mediated PCR (LM-PCR). A total of 114 retroviral integration sites were detected in vitro. 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Current analyses are focusing at the in vivo pattern of retroviral integration in DNA of TLCs obtained from transplanted patient’s TLCs. Therefore we developed a new high sensitive PCR method (HS-PCR), an improved LM-PCR to even detect minimal quantities of transduced DNA.

scholarly journals Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity

2019 ◽  
Vol 116 (42) ◽  
pp. 20969-20976 ◽  
Author(s):  
Yuanyan Tan ◽  
Athena H. Y. Chu ◽  
Siyu Bao ◽  
Duc Anh Hoang ◽  
Firaol Tamiru Kebede ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Genome Editing ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Genome Wide ◽  
Protospacer Adjacent Motif ◽  
Targeted Deep Sequencing ◽  
Targeting Accuracy ◽  
Target Effects

RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 776
Author(s):  
Shipra Kumari ◽  
Bashistha Kumar Kanth ◽  
Ju young Ahn ◽  
Jong Hwa Kim ◽  
Geung-Joo Lee
Keyword(s):  
Abiotic Stress ◽  
Biotic Stress ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Lilium Longiflorum ◽  
Biotic And Abiotic Stress ◽  
Biotic Stresses ◽  
Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

scholarly journals Methylation of rRNA as a host defense against rampant group II intron retrotransposition

Mobile DNA ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Justin M. Waldern ◽  
Dorie Smith ◽  
Carol Lyn Piazza ◽  
E. Jake Bailey ◽  
Nicholas J. Schiraldi ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Fold Increase ◽  
Group Ii Intron ◽  
In Vivo Experiments ◽  
Cellular Factors ◽  
Group Ii ◽  
Self Splicing ◽  
Native Host

Abstract Background Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. Results To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. Conclusions Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

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