Strategies for High-Efficiency Mutation Using the CRISPR/Cas System

Feng, Shi Ting; Wang, Zilong; Li, Aifang; Xie, Xin; Liu, Junjie; Li, Shuxuan; Li, Yalan; Wang, Baiyan; Hu, Lina; Yang, Lianhe; Guo, Tao

doi:10.3389/fcell.2021.803252

Cited by 10 publications

(3 citation statements)

References 264 publications

(292 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…By enhancing the specificity and precision of CRISPR/Cas9 delivery, biomaterials should be engineered to reduce off-target effects. This might include improving the design of the biomaterials or using improved CRISPR/Cas9 variants with increased selectivity (Feng et al, 2022;Iqbal et al, 2023). 2.…”

Section: Challengesmentioning

confidence: 99%

Biomaterials-mediated CRISPR/Cas9 delivery: recent challenges and opportunities in gene therapy

Dubey,

Mostafavi

2023

Front. Chem.

View full text Add to dashboard Cite

The use of biomaterials in delivering CRISPR/Cas9 for gene therapy in infectious diseases holds tremendous potential. This innovative approach combines the advantages of CRISPR/Cas9 with the protective properties of biomaterials, enabling accurate and efficient gene editing while enhancing safety. Biomaterials play a vital role in shielding CRISPR/Cas9 components, such as lipid nanoparticles or viral vectors, from immunological processes and degradation, extending their effectiveness. By utilizing the flexibility of biomaterials, tailored systems can be designed to address specific genetic diseases, paving the way for personalized therapeutics. Furthermore, this delivery method offers promising avenues in combating viral illnesses by precisely modifying pathogen genomes, and reducing their pathogenicity. Biomaterials facilitate site-specific gene modifications, ensuring effective delivery to infected cells while minimizing off-target effects. However, challenges remain, including optimizing delivery efficiency, reducing off-target effects, ensuring long-term safety, and establishing scalable production techniques. Thorough research, pre-clinical investigations, and rigorous safety evaluations are imperative for successful translation from the laboratory to clinical applications. In this review, we discussed how CRISPR/Cas9 delivery using biomaterials revolutionizes gene therapy and infectious disease treatment, offering precise and safe editing capabilities with the potential to significantly improve human health and quality of life.

show abstract

Section: Challengesmentioning

confidence: 99%

Biomaterials-mediated CRISPR/Cas9 delivery: recent challenges and opportunities in gene therapy

Dubey,

Mostafavi

2023

Front. Chem.

View full text Add to dashboard Cite

show abstract

“…The ability to scale up the use of CRISPR gene editing for testing VUS may help overcome these challenges. CRISPR technology has advanced rapidly with new approaches to improve efficiency constantly being developed (Feng et al, 2022;Skarnes et al, 2019). However, until the use of a CRISPR-based assay in cells becomes more practical for clinical laboratories, we recommend a variant-testing strategy that utilizes the previously calibrated CIMRA assay as a first test to identify those variants that clearly disrupt MMR function, as it seems unlikely that variants which severely affect biochemical activity in vitro would display proficient function in the cell (Drost et al, 2010(Drost et al, , 2019González-Acosta et al, 2020).…”

Section: Odds Of Pathogenicity Scoresmentioning

confidence: 99%

A calibrated cell‐based functional assay to aid classification of MLH1 DNA mismatch repair gene variants

et al. 2022

View full text Add to dashboard Cite

PURPOSE:Functional assays provide important evidence for classifying the disease significance of germline variants in the DNA mismatch repair genes. We sought to develop a cell-based approach for testing the function of variants of uncertain significance (VUS) in the MLH1 gene. METHODS:Using CRISPR gene editing, we knocked-in MLH1 VUS into the endogenous MLH1 loci in human embryonic stem cells. We examined their impact at the RNA and protein level, including their ability to maintain stability of microsatellite sequences and instigate a DNA damage response. We calibrated these assays by testing well-established pathogenic and benign control variants. RESULTS:Five VUS resulted in functionally abnormal protein, 15 VUS resulted in functionally normal protein, and one VUS showed mixed results. Furthermore, we converted the functional outputs into a single odds in favor of pathogenicity score for each VUS. CONCLUSION:Our CRISPR-based functional assay successfully models phenotypes observed in patients in a cellular context. Using this approach, we generated evidence for or against pathogenicity for utilization by variant classification expert panels. Ultimately, this information will assist in proper diagnosis and disease management for suspected Lynch syndrome patients.

show abstract

“…In contrast, making a specific, template-directed mutation at a DSB requires homology directed repair (HDR), which is active during the S-G2 phases of the cell cycle [ 6 , 7 , 8 ]. Since NHEJ predominates over HDR in most cells [ 5 ], knock-in editing is not very efficient, and, therefore, different strategies have been developed to improve HDR-mediated gene editing (reviewed in [ 9 ]).…”

Section: Introductionmentioning

confidence: 99%

Selecting for CRISPR-Edited Knock-In Cells

Reuven

Shaul

2022

IJMS

View full text Add to dashboard Cite

CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, “scarless” selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells.

show abstract

Strategies for High-Efficiency Mutation Using the CRISPR/Cas System

Cited by 10 publications

References 264 publications

Biomaterials-mediated CRISPR/Cas9 delivery: recent challenges and opportunities in gene therapy

Biomaterials-mediated CRISPR/Cas9 delivery: recent challenges and opportunities in gene therapy

A calibrated cell‐based functional assay to aid classification of MLH1 DNA mismatch repair gene variants

Selecting for CRISPR-Edited Knock-In Cells

Contact Info

Product

Resources

About