Engineered CRISPR Systems for Next Generation Gene Therapies

Pineda, Michael; Moghadam, Farzaneh; Ebrahimkhani, Mo R.; Kiani, Samira

doi:10.1021/acssynbio.7b00011

Cited by 29 publications

(23 citation statements)

References 140 publications

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“…Even though finding Cas9 variants with optimal activity at 20–24°C would be useful, our work shows that there can be advantages to working at temperatures that lower the activity of SpCas9. For example, off‐target mutations mediated by CRISPR/Cas9 can be lowered using temperature, and this finding will be important in the future when SpCas9 is used in clinical settings or for crop improvement (Pineda et al ., ).…”

Section: Discussionmentioning

confidence: 97%

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

et al. 2017

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The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9.

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Section: Discussionmentioning

confidence: 97%

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

et al. 2017

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“…T he discovery of gene editing and programmable genomic control by clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (crRNAs) and their CRISPR-associated (Cas) proteins [1][2][3][4][5] holds tremendous promise for future therapeutics and curing genetic diseases [6][7][8][9][10][11]. Despite their potential, CRISPR enzymes are not optimal for therapeutic applications [9,10,[12][13][14]. For example, they invariably suffer from unwanted ''off-target'' editing that can be difficult to predict, detect, or prevent [12,[15][16][17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9

Barkau

O'Reilly

Rohilla

et al. 2019

Nucleic Acid Therapeutics

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Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs and their associated effector (Cas) enzymes are being developed into promising therapeutics to treat disease. However, CRISPR-Cas enzymes might produce unwanted gene editing or dangerous side effects. Drug-like molecules that can inactivate CRISPR-Cas enzymes could help facilitate safer therapeutic development. Based on the requirement of guide RNA and target DNA interaction by Cas enzymes, we rationally designed small nucleic acid-based inhibitors (SNuBs) of Streptococcus pyogenes (Sp) Cas9. Inhibitors were initially designed as 2¢-O-methyl-modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9. Coupling anti-PAM and anti-tracr modules together was synergistic and resulted in high binding affinity and efficient inhibition of Cas9 DNA cleavage activity. Incorporating 2¢F-RNA and locked nucleic acid nucleotides into the anti-tracr module resulted in greater inhibition as well as dose-dependent suppression of gene editing in human cells. CRISPR SNuBs provide a platform for rational design of CRISPR-Cas enzyme inhibitors that should translate to other CRISPR effector enzymes and enable better control over CRISPR-based applications.

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“…There are some other CRISPR-related concerns such as runaway immune responses and spatiotemporal malfunctions, which result in off-targeting and the decrease of productivity, which have found solutions like development of engineered or split Cas enzymes in order to have more control on in vivo activity of CRISPR-Cas system [ 81 ].…”

Section: Crispr the Treasure Trove Of Gene-editing Technologiesmentioning

confidence: 99%

CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

et al. 2020

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Cancer immunotherapy has been emerged as a promising strategy for treatment of a broad spectrum of malignancies ranging from hematological to solid tumors. One of the principal approaches of cancer immunotherapy is transfer of natural or engineered tumor-specific T-cells into patients, a so called “adoptive cell transfer”, or ACT, process. Construction of allogeneic T-cells is dependent on the employment of a gene-editing tool to modify donor-extracted T-cells and prepare them to specifically act against tumor cells with enhanced function and durability and least side-effects. In this context, CRISPR technology can be used to produce universal T-cells, equipped with recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR), through multiplex genome engineering using Cas nucleases. The robust potential of CRISPR-Cas in preparing the building blocks of ACT immunotherapy has broaden the application of such therapies and some of them have gotten FDA approvals. Here, we have collected the last investigations in the field of immuno-oncology conducted in partnership with CRISPR technology. In addition, studies that have addressed the challenges in the path of CRISPR-mediated cancer immunotherapy, as well as pre-treatment applications of CRISPR-Cas have been mentioned in detail.

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Engineered CRISPR Systems for Next Generation Gene Therapies

Cited by 29 publications

References 140 publications

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress

Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9

CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

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