CRISPR Systems Suitable for Single AAV Vector Delivery

: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas gene editing is a revolutionary technology that can enable the correction of genetic mutations in vivo, providing great promise as a therapeutic intervention for inherited diseases. Adeno-associated viral (AAV) vectors are a potential vehicle for delivering CRISPR/Cas. However, they are restricted by their limited packaging capacity. Identifying smaller Cas orthologs that can be packaged, along with the required guide RNA elements, into a single AAV would be an important optimization for CRISPR/Cas gene editing. Expanding the options of Cas proteins that can be delivered by a single AAV not only increases translational application but also expands the genetic sites that can be targeted for editing. This review considers the benefits and current scope of small Cas protein orthologs that are suitable for gene editing approaches using single AAV vector delivery.

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CRISPR/Cas-based Diagnostics and Gene Therapy

BIO Integration ◽

10.15212/bioi-2020-0048 ◽

2021 ◽

Author(s):

Qiu Meiyu ◽

Li Pei

Keyword(s):

Gene Therapy ◽

Gene Editing ◽

Ex Vivo ◽

Point Of Care ◽

Cost Effective ◽

Blood Disorders ◽

Initial Stage ◽

Cas Gene ◽

Cas Proteins

Clustered regularly interspaced short palindromic repeats (CRISPR) technology, an easy, rapid, cost-effective, and precise gene-editing technique, has revolutionized diagnostics and gene therapy. Fast and accurate diagnosis of diseases is essential for point-of-care-testing (POCT) and specialized medical institutes. The CRISPR-associated (Cas) proteins system shed light on the new diagnostics methods at point-of-care (POC) owning to its advantages. In addition, CRISPR/Cas-based gene-editing technology has led to various breakthroughs in gene therapy. It has been employed in clinical trials for a variety of untreatable diseases, including cancer, blood disorders, and other syndromes. Currently, the clinical application of CRISPR/Cas has been mainly focused on ex vivo therapies. Recently, tremendous efforts have been made in the development of ex vivo gene therapy based on CRISPR-Cas9. Despite these efforts, in vivo CRISPR/Cas gene therapy is only in its initial stage. Here, we review the milestones of CRISPR/Cas technologies that advanced the field of diagnostics and gene therapy. We also highlight the recent advances of diagnostics and gene therapy based on CRISPR/Cas technology. In the last section, we discuss the strength and significant challenges of the CRISPR/Cas technology for its future clinical usage in diagnosis and gene therapy.

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Macrophage Modification Strategies for Efficient Cell Therapy

Cells ◽

10.3390/cells9061535 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1535 ◽

Cited By ~ 5

Author(s):

Anastasiya S. Poltavets ◽

Polina A. Vishnyakova ◽

Andrey V. Elchaninov ◽

Gennady T. Sukhikh ◽

Timur Kh. Fatkhudinov

Keyword(s):

Gene Expression ◽

Gene Editing ◽

Target Selection ◽

Model Systems ◽

Attractive Therapeutic Target ◽

Specific Alteration ◽

Inflammatory Phenotypes ◽

Vector Delivery

Macrophages, important cells of innate immunity, are known for their phagocytic activity, capability for antigen presentation, and flexible phenotypes. Macrophages are found in all tissues and therefore represent an attractive therapeutic target for the treatment of diseases of various etiology. Genetic programming of macrophages is an important issue of modern molecular and cellular medicine. The controllable activation of macrophages towards desirable phenotypes in vivo and in vitro will provide effective treatments for a number of inflammatory and proliferative diseases. This review is focused on the methods for specific alteration of gene expression in macrophages, including the controllable promotion of the desired M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes in certain pathologies or model systems. Here we review the strategies of target selection, the methods of vector delivery, and the gene editing approaches used for modification of macrophages.

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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.

10.1101/023366 ◽

2015 ◽

Cited By ~ 3

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.

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

10.1101/272724 ◽

2018 ◽

Cited By ~ 4

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.

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Faculty Opinions recommendation of AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726496362.793540445 ◽

2017 ◽

Author(s):

Yousin Suh

Keyword(s):

Gene Editing ◽

Retinal Cells ◽

Cas Gene

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Polyvalent Guide RNAs for CRISPR Antivirals

10.1101/2021.02.25.430352 ◽

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.

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

Science Advances ◽

10.1126/sciadv.aav7199 ◽

2019 ◽

Vol 5 (4) ◽

pp. eaav7199 ◽

Cited By ~ 56

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.

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Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus

Scientific Reports ◽

10.1038/s41598-019-54766-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Jill M. Goldstein ◽

Austin Valido ◽

Jordan P. Lewandowski ◽

Ryan G. Walker ◽

Melanie J. Mills ◽

...

Keyword(s):

Gene Editing ◽

Genetically Engineered ◽

Lymphoid Cells ◽

Null Alleles ◽

Gfp Expression ◽

Pcr Screening ◽

C Terminus ◽

Cas Gene

AbstractRecent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein (“GDF11-WE”) predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11WE allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.

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