A glance at genome editing with CRISPR–Cas9 technology

Barman, Antara; Deb, Bornali; Chakraborty, Supriyo

doi:10.1007/s00294-019-01040-3

Cited by 77 publications

(39 citation statements)

References 225 publications

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“…Non-specific cleavage of off-target sequences are a major concern with respect to the practical implementation of the CRISPR/Cas9 system [23,24]. In a deep sequencing analysis, more than 99% of the amplicons of potential off-target sites were made up of the WT sequence.…”

Section: Discussionmentioning

confidence: 99%

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

et al. 2020

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Background: Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pigto-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs. Results: We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets. Conclusions: We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.

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Section: Discussionmentioning

confidence: 99%

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

et al. 2020

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“…The clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein (Cas) system can provide versatile and promising tools against the growing challenge of multidrug resistance prevalence [10]. Among the different types of CRISPR-Cas systems, the CRISPR-Cas9 system is the most widely applied in gene editing [11]. By designing guide RNAs, the CRISPR-Cas9 system can kill targeted bacterial species possessing specific sequences in the involved bacterial community or destroy their antibiotic resistance genes, resensitizing them to antibiotics [12].…”

Section: Introductionmentioning

confidence: 99%

Novel Strategy to Combat Antibiotic Resistance: A Sight into the Combination of CRISPR/Cas9 and Nanoparticles

Wan

Draz

et al. 2021

Pharmaceutics

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Antibiotic resistance is a significant crisis that threatens human health and safety worldwide. There is an urgent need for new strategies to control multidrug-resistant (MDR) bacterial infections. The latest breakthrough in gene-editing tools based on CRISPR/Cas9 has potential application in combating MDR bacterial infections because of their high targeting ability to specifically disrupt the drug resistance genes that microbes use for infection or to kill the pathogen directly. Despite the potential that CRISPR/Cas9 showed, its further utilization has been hampered by undesirable delivery efficiency in vivo. Nanotechnology offers an alternative way to overcome the shortcomings of traditional delivery methods of therapeutic agents. Advances in nanotechnology can improve the efficacy and safety of CRISPR/Cas9 components by using customized nanoparticle delivery systems. The combination of CRISPR/Cas9 and nanotechnology has the potential to open new avenues in the therapy of MDR bacterial infections. This review describes the recent advances related to CRISPR/Cas9 and nanoparticles for antimicrobial therapy and gene delivery, including the improvement in the packaging and localizing efficiency of the CRISPR/Cas9 components in the NP (nanoparticle)/CRISPR system. We pay particular attention to the strengths and limitations of the nanotechnology-based CRISPR/Cas9 delivery system to fight nosocomial pathogens.We highlight the need for more scientific research to explore the combinatorial efficacy of various nanoparticles and CRISPR technology to control and prevent antimicrobial resistance.

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“…Many online and opensource tools exist to aid researchers in designing sgRNAs proximal to a PAM sequence to target their region of interest within the genome. Many of these tools have been gathered and organized by Barman et al [75].…”

Section: Harnessing Crispr For Genome Editingmentioning

confidence: 99%

Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Thompson

Sobol

Prakash

2021

Biology

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The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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A glance at genome editing with CRISPR–Cas9 technology

Cited by 77 publications

References 225 publications

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

Novel Strategy to Combat Antibiotic Resistance: A Sight into the Combination of CRISPR/Cas9 and Nanoparticles

Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

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