Gene and genome editing最新文献

As long read sequencing becomes more commonplace in genome analysis, the number of deletion variants associated with human traits and disease are anticipated to continue growing at a rate that may well outpace the increase in novel single nucleotide variant (SNV) discoveries. Such a paradigm shift will be met with an increased demand for gene editing technologies that enable functional analyses in a human model system such as induced pluripotent stem (iPS) cells. The outcome of gene editing is ultimately determined by cellular repair pathways and can be predicted by the surrounding DNA sequence. As multiple studies have revealed microhomology at the edges of natural deletion variants, eliciting microhomology mediated end joining (MMEJ) presents a reliable approach to create specific deletions. In this review, we discuss the shifting trends in human genome variant discovery, briefly review DNA repair processes and the associated prediction algorithms, demonstrate the utility of MMEJ in generating both loss- and gain-of-function alleles, and finally speculate on the impact these advances will have on the future of human functional genomics.

Nucleic acid amplification techniques (NATs) are frequently used in the molecular biology arena. Polymerase chain reaction (PCR) and its variants are one of the most popular NATs. The requirement of a sophisticated thermocycler and skilled technician for PCR limits its use in resource-limited laboratories and fields. Alternatively, the isothermal amplification technique can also deliver proficiency, simplicity, sensitivity, and fidelity without the need for the thermocycler. Several isothermal methods have been devised and newer concepts are also emerging. Some of them are loop-mediated isothermal amplification, whole genome amplification, rolling circle amplification, nucleic acid sequence-based amplification, and polymerase spiral reaction. These cost-effective, practicable, and easy-to-perform diagnostic assays are in current trends to identify pathogens, tumors, embryo sex, and genetically modified organisms. Isothermal amplification can also be used in microfluidic devices and point-of-need diagnostics. This review focuses on the development of common isothermal processes, their characteristics, and their acceptance over the PCR.

Antimicrobial resistance is a serious threat to the human population and might be responsible for the emergence as well as re-emergence of various infectious diseases. The staggard development of new antibiotics and the resistance acquired by the pathogens against the existing antibiotics is indeed a menace and has aggravated due to the ongoing pandemic. CRISPR-Cas systems, an inherent immune mechanism present in prokaryotes is one of the most popular tools that was first harnessed in 2014 for selective removal of genes responsible for antimicrobial resistance. Gradually gaining considerable momentum in the field of genetics, medicine, and biotechnology, CRISPR-Cas technologies have been rapidly utilized in gene editing in human cells, designing animal models for disease progression studies to develop insect-resistant crop varieties and repurpose inherent bacterial CRISPR-Cas systems for target specific elimination of pathogens. The main aim of this review is to discuss how the CRISPR-Cas systems have been utilized to produce new-generation antimicrobials, associated delivery vehicles and challenges, and the prospects of these powerful antimicrobials.

CRISPR-cas9 genome editing has received much attention in recent years due to its wide applications to treat various genetic disorders, cancer, and infectious diseases caused by harmful pathogens. Pseudomonas aeruginosa is one of the most prominent opportunistic pathogens that cause major concern in health care due to its antibiotic resistance. Quorum sensing inhibition is an effective means of treating this multidrug-resistant bacterial infection. In the present work, an in silico gene editing strategy was performed to knock out the LasR gene, responsible for regulating the expression of virulence-associated genes and biofilm formation in P. aeruginosa. To design appropriate guide RNA (gRNA) hits, the study explores four computational tools: ChopChop, Cas-Designer, Crispor, and Benchling which determine 18 gRNA hits out of 102 gRNAs, 39 hits out of 115, 6 hits out of 115, and 15 hits out of 115, respectively. About 19 hits that satisfy all the parameters mentioned in more than one tool were selected for further analysis. Thereafter, analysis of the 19 hits recommends gRNAs 1, 8, 14, 16, 17, and 19 as the top hits and subsequently, secondary structure analysis of the top hits using the RNAfold server ascertained gRNAs 1 and 16 as the best lead gRNAs. In addition, target-specific oligos and single guide RNAs (sgRNAs) for the selected leads were designed using the NEBiocalculator, followed by the in silico construction of the guide RNA expression vector using SnapGene software. However, the guide RNAs designed by computational methods need to be tested in vitro to determine their efficiency in knocking out the LasR gene.

Algae are oxygen-producing photosynthetic aquatic organisms. Algal biodiesels have attracted attention because these fuels are produced via photosynthesis, which assimilates CO₂. Algal biodiesels are sustainable, not competitive with food production, and have higher productivity compared with terrestrial plants. However, the production costs of algal biodiesels are much higher than those of fossil fuels. Therefore, improvement of algal lipid productivity is essential for the practical use of algal biodiesels. To achieve this, the application of genome-editing systems for the molecular breeding of algae is expected to generate “high-performance algae.” Here, we review the genome-editing technologies developed for the oleaginous microalgae, Nannochloropsis species, which are the most promising algae for producing algal-biodiesel feedstock. In this review, we discuss the development of genome-editing systems for gene disruption, transgene-free genome-editing systems, transcriptional regulation systems using nuclease-deficient Cas proteins, and the applications of genome editing in Nannochloropsis species.

CRISPR/Cas9 technology has been a powerful tool for gene editing in Drosophila, particularly for knocking in base-pair mutations or a variety of gene cassettes into endogenous gene loci. Among the Drosophila community, there has been a concerted effort to establish CRISPR/Cas9-mediated knock-in protocols that decrease the amount of time spent on molecular cloning. Here, we report the CRISPR/Cas9-mediated insertion of a ∼50 base-pair sequence into the ebony gene locus, using a linear double-stranded DNA (PCR product) donor template. By circumventing the cloning step of the donor template, our approach suggests the PCR product as a useful, alternative knock-in donor format.

Alteration of specific epigenetic marks might promote homology directed repair (HDR) during CRISPR-Cas9 genome editing. Testing several epigenetic inhibitors in a traffic light reporter assay, the histone methylation inhibitor 3DZNep showed a significant HDR promoting effect, while non-homologous end joining mediated repair was not significantly changed. This HDR promoting effect was largely independent of the target gene and its expression levels but showed a limited cell type specificity. HDR promotion was independent of the best described target of 3DZNep, the H3K27 methyltransferase EZH2, and of altered gene expression, but correlated partially with increased frequency of S/G2 cell cycle stage.

Genomic DNA is highly folded and is stored in the nucleus. In multicellular organisms, genomic DNA exhibits cell type-specific higher-order structures, including specific enhancer–promoter interactions, which have recently been shown to be relevant for cell type-specific regulation of gene expression. However, when the distances between specific enhancers and promoters were measured, cell-to-cell heterogeneity was unexpectedly large and, in some cases, did not correlate with the transcriptional state. These phenomena can be revealed by simultaneously visualizing specific genomic regions and the biological phenomena of interest. In this mini-review, I introduce methodologies for visualizing specific genomic DNA regions and provide detailed examples of how these techniques are being used to elucidate the mechanisms of transcriptional regulation.