Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

KEY MESSAGE: Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

CRISPR/Cas12a antifouling nanocomposite electrochemical biosensors enable amplification-free detection of Monkeypox virus in complex biological fluids.

The escalating global threat of infectious diseases, including monkeypox virus (MPXV), necessitates advancements in point-of-care diagnostics, moving beyond the constraints of conventional methods tethered to centralized laboratories. Here, we introduce multiple CRISPR RNA (crRNA)-based biosensors that can directly detect MPXV within 35 minutes without pre-amplification, leveraging the enhanced sensitivity and antifouling attributes of the BSA-based nanocomposite. Multiple crRNAs, strategically targeting diverse regions of the F3L gene of MPXV, are designed and combined to amplify Cas12a activation and its collateral cleavage of reporter probes. Notably, our electrochemical sensors exhibit the detection limit of 669 fM F3L gene without amplification, which is approximately a 15-fold improvement compared to fluorescence detection. This sensor also shows negligible changes in peak current after exposure to complex biological fluids, such as whole blood and serum, maintaining its sensitivity at 682 fM. This sensitivity is nearly identical to the conditions when only the F3L gene was present in PBS. In summary, our CRISPR-based electrochemical biosensors can be utilized as a high-performance diagnostic tool in resource-limited settings, representing a transformative leap forward in point-of-care testing. Beyond infectious diseases, the implications of this technology extend to various molecular diagnostics, establishing itself as a rapid, accurate, and versatile platform for detection of target analytes.

Interactive metabolic signatures of testicular testosterone with bilateral adrenalectomy in mice.

The hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes have reciprocal relationships with steroidogenesis regulation. However, the relationship between testicular steroids and defective glucocorticoid production under chronic stress remains unclear. Metabolic changes of testicular steroids in bilateral adrenalectomized (bADX) 8-week-old C57BL/6 male mice were measured using gas chromatography-mass spectrometry. Twelve weeks after surgery, testis samples were obtained from the model mice, which were divided into tap-water (n = 12) and 1 % saline (n = 24) supplementation groups, and their testicular steroid levels were compared with those of sham controls (n = 11). An increased survival rate with lower testicular levels of tetrahydro-11-deoxycorticosterone was observed in the 1 % saline group compared to both the tap-water (p = 0.029) and sham (p = 0.062) groups. Testicular corticosterone levels were significantly decreased in both tap-water (4.22 ± 2.73 ng/g, p = 0.015) and 1 % saline (3.70 ± 1.69, p = 0.002) groups compared to those in sham controls (7.41 ± 7.39). Testicular testosterone levels tended to increase in both bADX groups compared to those in the sham controls. In addition, increased metabolic ratios of testosterone to androstenedione in tap-water (2.24 ± 0.44, p < 0.05) and 1 % saline (2.18 ± 0.60, p < 0.05) mice compared to sham controls (1.87 ± 0.55) suggested increased production of testicular testosterone. No significant differences in serum steroid levels were observed. Defective adrenal corticosterone secretion and increased testicular production in bADX models revealed an interactive mechanism underlying chronic stress. The present experimental evidence suggests the crosstalk between the HPA and HPG axes in homeostatic steroidogenesis.

Evolution of CRISPR towards accurate and efficient mammal genome engineering.

The evolution of genome editing technology based on CRISPR (clustered regularly interspaced short palindromic repeats) system has led to a paradigm shift in biological research. CRISPR/Cas9-guide RNA complexes enable rapid and efficient genome editing in mammalian cells. This system induces double-stranded DNA breaks (DSBs) at target sites and most DNA breakages induce mutations as small insertions or deletions (indels) by non-homologous end joining (NHEJ) repair pathway. However, for more precise correction as knock-in or replacement of DNA base pairs, using the homology-directed repair (HDR) pathway is essential. Until now, many trials have greatly enhanced knock-in or substitution efficiency by increasing HDR efficiency, or newly developed methods such as Base Editors (BEs). However, accuracy remains unsatisfactory. In this review, we summarize studies to overcome the limitations of HDR using the CRISPR system and discuss future direction. [BMB Reports 2019; 52(8): 475-481].

Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy.

Adenine base editors (ABEs) composed of an engineered adenine deaminase and the Streptococcus pyogenes Cas9 nickase enable adenine-to-guanine (A-to-G) single-nucleotide substitutions in a guide RNA (gRNA)-dependent manner. Here we demonstrate application of this technology in mouse embryos and adult mice. We also show that long gRNAs enable adenine editing at positions one or two bases upstream of the window that is accessible with standard single guide RNAs (sgRNAs). We introduced the Himalayan point mutation in the Tyr gene by microinjecting ABE mRNA and an extended gRNA into mouse embryos, obtaining Tyr mutant mice with an albino phenotype. Furthermore, we delivered the split ABE gene, using trans-splicing adeno-associated viral vectors, to muscle cells in a mouse model of Duchenne muscular dystrophy to correct a nonsense mutation in the Dmd gene, demonstrating the therapeutic potential of base editing in adult animals.

Genome-wide target specificities of CRISPR RNA-guided programmable deaminases.

Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1-nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 ± 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.

Highly efficient RNA-guided base editing in mouse embryos.

Base editors (BEs) composed of a cytidine deaminase fused to CRISPR-Cas9 convert cytidine to uridine, leading to single-base-pair substitutions in eukaryotic cells. We delivered BE mRNA or ribonucleoproteins targeting the Dmd or Tyr gene via electroporation or microinjection into mouse zygotes. F0 mice showed nonsense mutations with an efficiency of 44-57% and allelic frequencies of up to 100%, demonstrating an efficient method to generate mice with targeted point mutations.

Optimization of reporter gene architecture for quantitative measurements of gene expression in the Drosophila embryo.

Quantitative assessment of gene regulation is critical for mathematical modeling of transcriptional systems for systems biology efforts. Enhancers, also termed cis-regulatory modules (CRMs), are the primary mediators of transcriptional regulation in higher eukaryotes; transcription factors binding to CRMs dictate the likelihood and frequency of promoter activation. To provide a suitable platform for in-depth CRM analysis, we adapted a targeted integration vector to compare action of basal promoters with diverse combination of TATA, Inr and DPE motifs, as well as a set of 3'-UTRs representative of those used in different reporter vectors. This "Honda" series of reporter gene vectors was activated by a regulatory element binding Dorsal and Twist activators suitable for transcription in the early Drosophila embryo. The diverse promoters functioned in a similar manner with minor quantitative differences, consistent with a lack of enhancer-promoter specificity. Constructs bearing SV40 3'-UTR sequences appeared to produce somewhat higher levels of mRNA. Confocal laser scanning microscopy revealed that the mRNA distribution produced by these constructs was punctate; this pattern appears to be dependent on 5'-UTR sequences, as an optimized vector including an alternate 5'-UTR produced a more even distribution, which may be preferable for quantitative imaging. This set of Honda vectors contains convenient sites for modification of basal promoter, 3' UTR, and enhancer, and will be useful for analysis of CRMs and quantitative studies of gene expression.