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Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.
CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.
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An inducer-independent, single-plasmid CRISPR-Cas9 system for genome editing in Bacillus species.
Advances in molecular biology tools are essential for streamlining and accelerating genetic engineering of cells across industrial and academic applications. While CRISPR-Cas improves genome editing efficiency, current systems have limitations and are often host specific, which restricts their versatility. This study describes a versatile CRISPR-Cas9 system for genome editing in industrially relevant Bacillus species. By adapting the well-established pJOE8999 vector-based CRISPR-Cas9 genome editing system, we constructed an inducer-independent, broad-host-range genome editing system. It maintains the benefits of low toxicity to the target cell and the cloning host as well as the ease to use of a single-plasmid CRISPR-Cas9 system. We utilized the constitutive Sigma70-type promoter from the conserved veg gene of Bacillus, to develop and test the suitability of promoter variants of different strengths for Cas9 expression. Successful gene deletions in three different Bacillus species demonstrated the versatility of the modified system for this industrially important genus. This was further confirmed by the integration of a reporter gene fusion and the introduction of a single point mutation in the genome of Bacillus licheniformis. This one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time. KEY POINTS: • Editing and screening of promoter variants for balanced Cas9 expression in Bacillus. • Development of a versatile inducer-independent, single-plasmid CRISPR-Cas-based system. • Verification of the modified CRISPR-based system for genome editing in different Bacilli.
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Amide Internucleoside Linkages Are Well Tolerated in Protospacer Adjacent Motif-Distal Region of CRISPR RNAs.
The development of CRISPR-Cas9 mediated gene editing technology is revolutionizing molecular biology, biotechnology, and medicine. However, as with other nucleic acid technologies, CRISPR would greatly benefit from chemical modifications that optimize delivery, activity, and specificity of gene editing. Amide modifications at certain positions of short interfering RNAs have been previously shown to improve their RNAi activity and specificity, which motivated the current study on replacement of selected internucleoside phosphates of CRISPR RNAs with amide linkages. Herein, we show that amide modifications did not interfere with CRISPR-Cas9 activity when placed in the protospacer adjacent motif (PAM) distal region of CRISPR RNAs. In contrast, modification of the seed region led to a loss of DNA cleavage activity at most but not all positions. These results are encouraging for future studies on amides as backbone modifications in CRISPR RNAs.
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DNA-Free Genome Editing via Ribonucleoprotein (RNP) Delivery of CRISPR/Cas in Lettuce.
CRISPR/Cas9 nuclease system is getting popular in precise genome editing of both eukaryotic and prokaryotic systems due to its accuracy, programmability, and relative ease of use. CRISPR/Cas systems can be delivered into live cells via plasmid DNA, RNA, and ribonucleoprotein (RNP). Of these, the RNP method is of special interest due to enzymatic action in shorter time and controllability over their activity. In addition, because RNP does not involve DNA, none of unwanted DNA footprints are left in the host genome. Previously, we demonstrated that plant protoplasts can be transfected with functional RNPs and the whole plants can be regenerated from an engineered protoplast. Relative to the published methods, the revised protocols described here should help increase the success rate of whole plant regeneration by reducing damages to the naked protoplast cells.
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Building the Class 2 CRISPR-Cas Arsenal.
Adaptation of CRISPR-Cas9 for genome-editing applications has revolutionized biomedical research. New single-component effector CRISPR systems are emerging from the bioinformatics pipeline. How can we best harness their power? Three new studies will no doubt facilitate this transition by generating the C2c1 and C2c2 structure snapshots in different functional states.
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Efficient genome engineering in Mycolicibacterium neoaurum using Cas9 from Streptococcus thermophilus.
Non-pathogenic mycobacteria, including Mycolicibacterium neoaurum, can directly utilize phytosterols for large-scale industrial production of steroid medicine intermediates due to their natural steroid metabolism pathway. The targeted genetic modification of M. neoaurum is conducive to the selection of high-yield engineering bacteria with high-value-added product, such as Pregnadien-20-carboxylic acid (PDC), which is an important precursor for synthesizing some corticosteroids. Based on heterologous type II CRISPR/sth1Cas9 system, a simple strategy was developed to genetic engineer M. neoaurum genome. Here, a customizable plasmid tool pMSC9 was constructed from pMV261 with integration of sth1Cas9 protein and corresponding sgRNA scaffold. Subsequently, the pMSC9 was inserted with spacer sequences corresponding to different targeted genes, generating editing plasmids, and then transformed into M. neoaurum. As a result, the targeted genes were introduced with DNA double stand breaks (DSBs) by CRISPR/sth1Cas9 system and then repaired by innate non-homologous end-joining (NHEJ) mechanism. Finally, editing plasmids were cured from correctly edited M. neoaurum mutants by means of no resistance cultivation, and the resulting mutant deleting the one target gene was used as the host to which another target gene could be deleted via the same process. This study demonstrated that the CRISPR/sth1Cas9 tool allowed M. neoaurum strains to be rapidly edited. And the editing mode of CRISPR/sth1Cas9 system indicated that this tool was an important supplement to the gene editing toolbox of M. neoaurum.
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CRISPR/Cas9-mediated germline mutagenesis in the subsocial parasitoid wasp, Sclerodermus guani.
The ectoparasitoid wasp Sclerodermus guani (Hymenoptera: Bethylidae), as a subsocial insect, is widely applied in biological control against beetle vectors of pine wood nematodes. Despite significant advances in behavioural research, functional genetics in S. guani remains underdeveloped due to the absence of efficient gene manipulation tools. In this study, we employed CRISPR-mediated mutagenesis to achieve germline gene knockout targeting the eye pigment-associated gene kynurenine 3-monooxygenase (KMO). Phylogenetic analysis revealed that S. guani KMO shares a close relationship with its homologue in Prorops nasuta (Hymenoptera: Bethylidae). Two single-guide RNAs (sgRNAs), coupled with Cas9 protein with and without nuclear localisation signal (NLS) were tested. Both sgRNAs induced specific in vitro DNA cleavage and in vivo heritable indels at the target genomic loci. Homozygous null mutant females and males exhibit a white-eye phenotype, which was identified during pupal stage. Optimal editing efficiency in vivo was achieved using the Cas9-NLS variant. Given the complication of germline gene editing in eusocial Hymenopterans, the application of CRISPR in the subsocial parasitoid wasp S. guani provides an accessible research platform for the molecular evolution of insect sociality.
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Innovative genetic scissor strategies and their applications in cancer treatment and prevention: CRISPR modules and challenges.
There are lots of gene editing tools for targeting genome sequences. Some are almost known, and most are a complete mystery and undiscovered. CRISPR/Cas editing tools have brought about a major revolution in medicine. Researchers have shown that CRISPR can modify DNA much more accurately, economically and easily than previous methods. CRISPR has proven itself effective for the deletion, replacement and insertion of DNA fragments into cell types, tissues and organisms. Recently, combining CRISPR/Cas with factors (transcription factors/repressors, exonucleases, endonucleases, transposons, caspase, fluorescent proteins, oxidoreductive enzymes, DNA/RNA polymerases), and elements (aptamers, barcodes, fluorescent probes, Trigger) have provided genome, transcriptome, proteome and epigenome modification. These modules are being investigated for cancer prevention and therapy and this review focuses on such innovative combinations that hopefully will become a clinical reality in the near future.
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One-Step Assembly of a PRRSV Infectious cDNA Clone and a Convenient CRISPR/Cas9-Based Gene-Editing Technology for Manipulation of PRRSV Genome.
Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes.
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Electroporation of sheep zygotes as an alternative to microinjection for the generation of CRISPR/Cas genome edited models.
Zygote microinjection is considered the most suitable technique to introduce CRISPR/Cas9 reagents for efficient genome editing in livestock. In this study, zygote electroporation was evaluated as an alternative to microinjection for CRISPR/Cas9-mediated genome editing in sheep. Four experiments were conducted on 3548 cumulus-oocyte complexes. Acid Tyrode's solution (AT) was used to partially degrade the zona pellucida (ZP) to improve reagent entry, resulting in ZP thinning with longer AT exposure (P < 0.05). Although early embryo development was impaired by AT exposure (P < 0.05), blastocyst rates were similar across all groups by day 8. Electroporation conditions were optimized by testing pulse length (1 or 3 ms), with the best results from 6 pulses of 20 V for 3 ms with AT during 60 s. Electroporation with 500 ng/μL Cas9 and 300 ng/μL sgRNA with AT during 60 s achieved a 38.5 % mutation rate. When compared with conventional microinjection, electroporation had higher developmental rates but a lower mutation rate (21.4 % vs. 60.0 %; P < 0.05). These findings suggest that electroporation is a viable, cost-effective technique for genome editing in sheep. Nevertheless, further research will be required to fine-tune electroporation conditions and enhance efficiency in terms of mutation rate.
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