Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance

The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes.

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“…This system also demonstrated higher editing efficacy compared to unmodified Cas9/sgRNA complexed with conventional lipids. 79 …”

Section: Physical and Non-viral Delivery Of Crispr-cas9mentioning

confidence: 99%

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

Liu

zhang

Líu

et al. 2017

Journal of Controlled Release

454

399

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“…61 In addition, owing to the delivery system that acts in higher organisms, RNA-guided nucleases could enable us to modulate the prevalence of specific genes such as antibiotic resistance genes and virulence determinants in wild-type populations. 87 The Cas9 nuclease targeting specific DNA sequences of bacterial pathogens and antibiotic resistance gene are delivered to microbial populations using polymer-derivatized CRISPR nanocomplexes, 36 bacteria carrying plasmids transmissible by conjugation, 61,88 and/or bacteriophages 60,61 (Figure 2). Gomma et al 62 used the typed I-E CRISPR-Cas system of E. coli, encoding six cas genes in two operons including casABCDE and cas3, 53 to specific and essential sequences in the genomes of different sequences.…”

Section: Crispr-cas System Neutralizing Antibiotic-resistant Genesmentioning

confidence: 99%

“…In addition, they demonstrated that the CRISPR-Cas system could discriminate between resistant and susceptible strains, which found that programmed gyrA phagemid was specifically cytotoxic only for chromosomal gyrA mutations that are responsible to quinolone-resistant E. coli 94 and not cytotoxic for isogenic strains with the wild type gyrA gene. 61 In another study, Kang et al 36 introduced a nonviral delivery method based on a Cas9-nanocomplex, which used Cas9, sgRNA targeting mecA, and a cationic polymer, known as branched polyethyleneimine (bPEI). The bPEI is one of the most commonly used carriers for gene delivery 95,96 and was used as the carrier for packaging sgRNA, which can enhance Cas9 delivery into MRSA strains.…”

Section: Crispr-cas System Neutralizing Antibiotic-resistant Genesmentioning

confidence: 99%

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<p>How CRISPR-Cas System Could Be Used to Combat Antimicrobial Resistance</p>

Gholizadeh¹,

Köse²,

Dao³

et al. 2020

IDR

109

101

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Rapid emergence of antibiotic-resistant bacteria has made it harder for us to combat infectious diseases and to develop new antibiotics. The clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR-Cas) system, as a bacterial adaptive immune system, is recognized as one of the new strategies for controlling antibioticresistant strains. The programmable Cas nuclease of this system used against bacterial genomic sequences could be lethal or could help reduce resistance of bacteria to antibiotics. Therefore, this study aims to review using the CRISPR-Cas system to promote sensitizing bacteria to antibiotics. We envision that CRISPR-Cas approaches may open novel ways for the development of smart antibiotics, which could eliminate multidrug-resistant (MDR) pathogens and differentiate between beneficial and pathogenic microorganisms. These systems can be exploited to quantitatively and selectively eliminate individual bacterial strains based on a sequence-specific manner, creating opportunities in the treatment of MDR infections, the study of microbial consortia, and the control of industrial fermentation.

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“…The limitations associated with both viral delivery and physical delivery can be addressed using non-viral delivery systems such as lipid nanoparticles (23)(24)(25)(26)(27), DNA nanoclew (28), gold nanoparticles (29,30), as well as chemically conjugating Cas9 protein with polymers (31) and cell penetrating peptides (32). Furthermore, direct delivery of Cas9-sgRNA ribonucleoprotein (RNP) is being considered as a promising therapeutic strategy.…”

Section: Introductionmentioning

confidence: 99%

CRISPR delivery particles for developing therapeutic strategies in metabolic disease

Shen

Cohen

Nicoloro

et al. 2018

Preprint

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RNA-guided engineered nucleases derived from a prokaryotic adaptive immune system known as CRISPR-Cas represent a promising platform for gene deletion and editing. As a therapeutic approach, direct delivery of Cas9 protein and guide RNA could circumvent the safety problems associated with plasmid delivery and therefore represents an attractive tool for genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fat oxidation and secretion of bioactive factors through a "browning" process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed novel CRISPR delivery particles, denoted CriPs, composed of nano-size complexes of Cas9 protein and single guide (sg)RNA, coated with an amphipathic peptide called Endo-Porter that mediates entry into cells.Efficient CRISPR-Cas9 mediated gene deletion of ectopically expressed Green fluorescent protein (GFP) by CriPs was achieved in multiple cell types including a macrophage cell line, primary macrophages and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP expressing mice following intraperitoneal injection of CriPs containing sgRNA targeting Gfp. Furthermore, the disruption of the Nrip1 gene in white adipocytes by CriPs enhanced adipocyte "browning" with a marked increase of UCP1 expression. Deletion of Nrip1 by CriPs did not produce detectable off-target effects. Thus CriPs represent a novel CRISPR delivery system for Cas9 and sgRNA that is effective for ablating targeted gene products in cultured cells and in vivo, and provide a potential therapeutic strategy for metabolic disease.

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Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance

Cited by 156 publications

References 48 publications

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

<p>How CRISPR-Cas System Could Be Used to Combat Antimicrobial Resistance</p>

CRISPR delivery particles for developing therapeutic strategies in metabolic disease

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