scholarly journals Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

2019 ◽  
Author(s):  
Jacob Lamberth ◽  
Laura Daley ◽  
Pachai Natarajan ◽  
Stanislav Khoruzhenko ◽  
Nurit Becker ◽  
...  
Keyword(s):  
Cell Culture ◽  
Genome Editing ◽  
Plasmid Dna ◽  
High Efficiency ◽  
High Specificity ◽  
Human Cells ◽  
Dna And Rna ◽  
Genome Modification ◽  
Delivery Technology ◽  
Target Effects

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

scholarly journals CRISPR-Cas9 genome editing in human cells works via the Fanconi Anemia pathway

2017 ◽  
Author(s):  
Chris D Richardson ◽  
Katelynn R Kazane ◽  
Sharon J Feng ◽  
Nicholas L Bray ◽  
Axel J Schäfer ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Fanconi Anemia ◽  
High Efficiency ◽  
Genetic Diseases ◽  
Dermal Fibroblasts ◽  
Human Cells ◽  
Individual Gene ◽  
Break Site ◽  
Repair Pathways

AbstractCRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype and/or transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

scholarly journals Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects

2021 ◽  
Vol 12 ◽  
Author(s):  
Yufan Xu ◽  
Xiaorong Peng ◽  
Yanghao Zheng ◽  
Changzhong Jin ◽  
Xiangyun Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Lentiviral Vector ◽  
Host Cells ◽  
Preliminary Evaluation ◽  
Major Barrier ◽  
Editing Efficiency ◽  
Latent Hiv ◽  
Hiv 1 ◽  
Target Effects

Viral DNA integrated in host cells is a major barrier to completely curing HIV-1. However, genome editing using the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has the potential to eradicate HIV-1. The present study aimed to use a lentiviral vector-based CRISPR/Cas9 system combined with dual-small/single guide RNAs (sgRNAs) to attack HIV-1 DNA in the latency reactivation model J-Lat 10.6 cell line and to assess off-target effects using whole-genome sequencing (WGS). We designed 12 sgRNAs targeting HIV-1 DNA, and selected high-efficiency sgRNAs for further pairwise combinations after a preliminary evaluation of the editing efficiency. Three combinations of dual-sgRNAs/Cas9 with high editing efficiency were screened successfully from multiple combinations. Among these combinations, the incidences of insertions and deletions in the sgRNA-targeted regions reached 76% and above, and no credible off-target sites were detected using WGS. The results provided comprehensive basic experimental evidence and methodological recommendations for future personalized HIV-1 treatment using CRISPR/Cas9 genome editing technology.

scholarly journals Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants

2019 ◽  
Vol 20 (15) ◽  
pp. 3719 ◽  
Author(s):  
Zahra Hajiahmadi ◽  
Ali Movahedi ◽  
Hui Wei ◽  
Dawei Li ◽  
Yasin Orooji ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Short Palindromic Repeat ◽  
External Forces ◽  
Time And Energy ◽  
Reduced Time ◽  
Target Effects

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

scholarly journals TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity

2014 ◽  
Vol 42 (10) ◽  
pp. 6762-6773 ◽  
Author(s):  
Claudio Mussolino ◽  
Jamal Alzubi ◽  
Eli J. Fine ◽  
Robert Morbitzer ◽  
Thomas J. Cradick ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Specificity ◽  
Human Cells ◽  
Low Cytotoxicity

scholarly journals No observable guide-RNA-independent off-target mutation induced by prime editor

2021 ◽  
Author(s):  
Runze Gao ◽  
Zhi-Can Fu ◽  
Xiangyang Li ◽  
Ying Wang ◽  
Jia Wei ◽  
...  
Keyword(s):  
Transcriptome Sequencing ◽  
High Specificity ◽  
Human Cells ◽  
Whole Genome ◽  
Guide Rna ◽  
Genome Wide ◽  
Target Sites ◽  
Whole Transcriptome Sequencing ◽  
Whole Transcriptome ◽  
Target Effects

Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.

High-efficiency transfection of small RNAs and plasmid DNA in human cells

2007 ◽  
Author(s):  
Graziano Martello ◽  
Michelangelo Cordenonsi ◽  
Stefano Piccolo
Keyword(s):  
Plasmid Dna ◽  
Small Rnas ◽  
High Efficiency ◽  
Human Cells

scholarly journals Reducible Branched Ester-Amine Quadpolymers (rBEAQs) Codelivering Plasmid DNA and RNA Oligonucleotides Enable CRISPR/Cas9 Genome Editing

2019 ◽  
Vol 11 (11) ◽  
pp. 10472-10480 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
Katie Sanders ◽  
Jordan J. Green
Keyword(s):  
Genome Editing ◽  
Plasmid Dna ◽  
Dna And Rna

scholarly journals The new genomic editing system (CRISPR)

2019 ◽  
Author(s):  
Mehrdad Talebi ◽  
Mohammad Yahya Vahidi Mehrjardi
Keyword(s):  
Genetic Engineering ◽  
Genome Editing ◽  
High Specificity ◽  
Repair System ◽  
End Joining ◽  
Genetic Manipulations ◽  
Dna And Rna ◽  
A Cell ◽  
Non Homologous End Joining ◽  
Genomic Editing

Over the past decades, progression in genetic element manipulation, and consequently, the treatment of diseases has been remarkable. It is worth noting that these genetic manipulations perform at different levels, including DNA and RNA. The earlier genomic editing techniques, including MN, ZFN , TALEN , performing their functions by creating double-stranded breaks (DSBs), and after breakage, the cell tries to repair the breakage through two systems, homologous recombination and non-homologous end joining. CRISPR/Cas technology has been discovered recently, and has become the most widely used genome-editing tool, mainly due to its capabilities and those added to this through the genetic engineering. In this study, we aimed to introduce a variety of CRISPR classes in the elementary parts, and then the modified CRISPR systems developed to increase the efficiency and specificity of the system and provide acceptable results will be introduced. In this study, for three months in the fall and winter, Pubmed and Web of science sites searched for keywords such as CRISPR, Types of CRISPR, gRNA, Cas9, and CRISPR-Cas9 nickase that eventually resulted in about four hundred Sixty-one articles, and some of these articles after closer study, reviewed in this article. Genetic engineering techniques have successfully transformed this system into the most efficient genome editing tool in recent years. Researchers are working on a system to treat various diseases by resolving problems such as high specificity, cutting off non-target sites, how to move to a cell, and setting up a proper repair system.

scholarly journals CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1649
Author(s):  
Hao Cheng ◽  
Feng Zhang ◽  
Yang Ding
Keyword(s):  
Genome Editing ◽  
Plasmid Dna ◽  
Biomedical Applications ◽  
Viral Vectors ◽  
Recent Progress ◽  
Disease Modeling ◽  
Gene Correction ◽  
Cas9 Protein ◽  
Clinical Potential ◽  
Delivery Technology

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing.

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