- Award ID(s):
- NSF-PAR ID:
- Date Published:
- Journal Name:
- Horticulture Research
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.more » « less
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pcsk9, apoc3, and angptl3would improve cardiovascular health by lowering blood cholesterol and triglycerides, the lipoMSN is used to deliver a combination of RNPs targeting these genes. When targeting a single gene, the lipoMSN achieved a 54% gene‐editing efficiency, besting the state‐of‐art Lipofectamine CRISPRMax. For multiplexing, lipoMSN maintained significant gene‐editing at each gene target despite reduced dosage of target‐specific RNP. By delivering combinations of targeting RNPs in the same nanoparticle, synergistic effects on lipid metabolism are observed in vitro and vivo. These effects, such as a 50% decrease in serum cholesterol after 4 weeks of post‐treatment with lipoMSN carrying both pcsk9and angptl3‐targeted RNPs, could not be reached with a single gene‐editing approach. Taken together, this lipoMSN represents a versatile platform for the development of efficient, combinatorial gene‐editing therapeutics.
CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the moss
Physcomitrella patens. However, CRISPR‐Cas9‐mediated genome editing in P. patenswas shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing in P. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping.
CRISPR/Cas technology is increasingly being used as a common methodology in many cancer biology studies due to the ease and convenience of the technique. Precise editing of genomic DNA has been achieved upon repair of CRISPR-induced DNA double-strand breaks (DSBs) by homologous recombination (HR). HR repairs DNA DSBs with high fidelity and therefore, deficiencies in HR result in genome instability. These deficiencies have been demonstrated in many cancers. RAD51-dependent HR is a very important pathway for repairing DSBs. Previous studies have shown that genome editing using CRISPR technology relies on the repair of site-specific DNA DSBs induced by the RNA-guided Cas9 endonuclease. Furthermore, previous studies have shown that the efficiency of CRISPR-mediated HR can be improved by the stimulation of HR promoting factors, such as the RAD51 recombinase. Despite the ease and efficient use the CRISPR/Cas technology for genome editing, one limitation is the potential occurrence of associated off-target effects. If CRISPR technology is planned to be used to target cancer cells with defective HR capabilities, will off-target mutations be likely to occur? In order to answer this question, a system was developed in Saccharomyces cerevisiae using green fluorescent protein (GFP) as a reporter to identify off-target CRISPR-induced DSBs. This study set out to test the number of off-target DSBs that could be introduced by CRISPR-induced genome editing in a RAD51-deficient HR model. We were curious whether loss of RAD51-dependent HR would increase the abundance of off-target CRISPR-induced DSBs in mutant yeast strains as compared to those with a functioning HR-dependent DNA repair pathway. Preliminary findings using this system will be presented.more » « less
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