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Genome editing of pufferfish saxitoxin- and tetrodotoxin-binding protein type 2 in Takifugu rubripes.
The pufferfish saxitoxin- and tetrodotoxin-binding protein 2 (PSTBP2), which is involved in toxin accumulation, was knocked out in Takifugu rubripes embryos by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome-editing technology. Treating the embryos with one of two single-guide RNA (sgRNA) resulted in mutation rates of 57.1% and 62.5%, respectively, as estimated using a heteroduplex mobility assay at 3 days postfertilization. Both sgRNAs might induced frameshift mutations that knocked out the T. rubripes PSTBP2.
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Opioid Addiction, Genetic Susceptibility, and Medical Treatments: A Review.
Opioid addiction is a chronic and complex disease characterized by relapse and remission. In the past decade, the opioid epidemic or opioid crisis in the United States has raised public awareness. Methadone, buprenorphine, and naloxone have proven their effectiveness in treating addicted individuals, and each of them has different effects on different opioid receptors. Classic and molecular genetic research has provided valuable information and revealed the possible mechanism of individual differences in vulnerability for opioid addiction. The polygenic risk score based on the results of a genome-wide association study (GWAS) may be a promising tool to evaluate the association between phenotypes and genetic markers across the entire genome. A novel gene editing approach, clustered, regularly-interspaced short palindromic repeats (CRISPR), has been widely used in basic research and potentially applied to human therapeutics such as mental illness; many applications against addiction based on CRISPR are currently under research, and some are successful in animal studies. In this article, we summarized the biological mechanisms of opioid addiction and medical treatments, and we reviewed articles about the genetics of opioid addiction, the promising approach to predict the risk of opioid addiction, and a novel gene editing approach. Further research on medical treatments based on individual vulnerability is needed.
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Implementation of the CRISPR-Cas9 system in fission yeast.
Application of the CRISPR-Cas9 genome editing system in the model organism Schizosaccharomyces pombe has been hampered by the lack of constructs to express RNA of arbitrary sequence. Here we present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA. Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%. The rrk1 CRISPR-Cas9 method enables rapid and efficient genome manipulation and unlocks the CRISPR toolset for use in fission yeast.
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Shaping the future of date palm (Phoenix dactylifera) through new genetic improvement strategies.
Conventional breeding of date palm (Phoenix dactylifera ) is inherently challenging due to its long generation time, dioecious nature, and high genetic heterogeneity. However, current developments in genomics and molecular biology offer promising avenues for accelerating breeding programs, particularly through high-throughput technologies including functional genomics. This article reviews genomic tools such as like CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9) that may bring significant changes in date palm breeding. The CRISPR-Cas9 system enables scientists to accurately target genomic regions, which helps enhance breeding accuracy by adding advantageous traits and eliminating unfavorable genes through precision editing. Transcriptome and metabolome analyses have also explained the regulation of thousands of differentially expressed genes (DEGs) and metabolic pathways under environmental stress. These studies contribute to enhance the knowledge of stress tolerance mechanisms, which include the secondary metabolic process of flavonoids. Genomic studies illustrating single nucleotide polymorphism (SNP)-based diversity between cultivars from north African and the Arabian Gulf provide new genetic resources for selective breeding. The work relates genome-wide association studies (GWAS) and miRNA profiling to elucidate key regulatory networks involved in fruit development and stress resilience. The integration of such advanced technologies, especially the CRISPR-Cas9 system, is revolutionizing the landscape of date palm breeding, opening new avenues for accelerated development of superior cultivars that meet the needs of modern agriculture.
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Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides.
Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.
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Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format.
The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.
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Functional Analysis of Nuclear Estrogen Receptors in Zebrafish Reproduction by Genome Editing Approach.
Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII), and esr2b (ERβI), by the genome-editing technology clustered regularly interspaced short palindromic repeats and its associated nuclease (CRISPR/Cas9). Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double, and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male. In addition, the current study also revealed a unique role for Esr2a in follicle cell proliferation and transdifferentiation, follicle growth, and chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a, and esr2b in fish reproduction.
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Development of Knockout Cardiac Muscle Cell Lines Using Integrase-Deficient Lentivirus-Mediated CRISPR/Cas9 Gene Editing.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology is a highly efficient genome editing tool that can genetically disrupt genes and genetic elements, making it a timely, cost-effective, and powerful tool for studying gene function. The success of gene editing depends on the ability to introduce CRISPR components, including guide RNA (gRNA) and Cas9 nuclease, into the target cell, which is challenging in numerous difficult-to-transfect cell types, such as cardiomyocytes. Lentiviral vectors (LVs) are among the primary delivery methods for the CRISPR/Cas9 system as they can stably maintain robust expression in various dividing and non-dividing cells. However, stably integrated LVs consistently express CRISPR/Cas9 components at high levels, rendering them susceptible to off-target effects. New-generation integrase-deficient LV (IDLV) offers an attractive alternative approach for delivering CRISPR/Cas9 components. This study constructed transient receptor potential cation channel mucolipin subfamily member 1 gene knockout models in H9C2 cell lines using IDLVs. Strategies for gRNA design and screening, the IDLV packaging process, CRISPR delivery, and knockout validation are outlined. These protocols will assist researchers in the application of CRISPR technology to study gene function in mammalian cells.
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Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System.
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9 construct was transformed into maize protoplasts, and indel (insertion and deletion) mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sgRNA-Cas9 constructs were transformed into protoplasts followed by sequencing the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM (protospacer adjacent motif) or having more than two mismatches. Together, our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize.
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Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection.
Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.
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