Recapitulating familial hypercholesterolemia in a mouse model by knock-in patient-specific LDLR mutation.

Familial hypercholesterolemia (FH) is one of the most prevalent monogenetic disorders leading to cardiovascular disease (CVD) worldwide. Mutations in Ldlr, encoding a membrane-spanning protein, account for the majority of FH cases. No effective and safe clinical treatments are available for FH. Adenine base editor (ABE)-mediated molecular therapy is a promising therapeutic strategy to treat genetic diseases caused by point mutations, with evidence of successful treatment in mouse disease models. However, due to the differences in the genomes between mice and humans, ABE with specific sgRNA, a key gene correction component, cannot be directly used to treat FH patients. Thus, we generated a knock-in mouse model harboring the partial patient-specific fragment and including the Ldlr W490X mutation. LdlrW490X/W490X mice recapitulated cholesterol metabolic disorder and clinical manifestations of atherosclerosis associated with FH patients, including high plasma low-density lipoprotein cholesterol levels and lipid deposition in aortic vessels. Additionally, we showed that the mutant Ldlr gene could be repaired using ABE with the cellular model. Taken together, these results pave the way for ABE-mediated molecular therapy for FH.

Structural diversity and biological role of the 5' untranslated regions of picornavirus.

The genomic arrangement of most picornavirus of the Picornaviridae family shares a similar monocistronic genomic pattern and a defining organizational feature. A defining feature of picornavirus is the presence of evolutionarily conserved and highly-structured RNA elements in untranslated regions (UTRs) at the genome' 5'and 3' ends, essential for viral replication and translation. Given the diversity and complexity of RNA structure and the limitations of molecular biology techniques, the functional characterization and biological significance of UTRs remain to be fully elucidated, especially for 5' UTR. Here, we summarize the current knowledge of the 5' UTR of picornavirus. This review focuses on the structural characterization and the biological function of the RNA secondary and tertiary structures in the 5' UTR of picornavirus. Understanding the role of the 5' UTR of picornavirus can provide a deep insight into the viral replication cycle and pathogenic mechanisms.

Engineered Staphylococcus auricularis Cas9 with high-fidelity.

CRISPR-Cas9 is a versatile gene editing tool with a broad application of basic research and clinical therapeutics. However, the potential impact caused by off-target effects remains a critical bottleneck. The small Cas9 ortholog from Staphylococcus auricularis (SauriCas9) was identified, which recognizes a 5'-NNGG-3' protospacer adjacent motif (PAM), exhibiting high activity for genome editing. Recently, we also reported enhanced-fidelity Staphylococcus aureus Cas9 (efSaCas9), which harbors a single mutation N260D. Protein sequence alignment revealed that SauriCas9 has 62.4% sequence identity with SaCas9. Because SauriCas9 is more flexible in recognizing the target sequence with PAM of 5'-NNGG-3' than SaCas9 of 5'-NNGRRT-3' PAM, we sought to test whether key mutation(N260D) or adjacent residue mutation in efSaCas9 can be appliable to SauriCas9. With this concept, two engineered SauriCas9 variants (SauriCas9-HF1, harboring the N269D mutation; SauriCas9-HF2, harboring the D270N mutation) dramatically improved targeting specificity by targeted deep sequencing and GUIDE-seq. At certain sites, reduced off-target effects (approximately 61.6- and 111.9-fold improvements) of SauriCas9-HF2 compared with wild-type SauriCas9 were observed. Overall, two identified SauriCas9 variants (SauriCas9-HF1 and SauriCas9-HF2) expand the utility of the CRISPR toolkit for research and therapeutic applications.

Green Fluorescent Protein Tagged Polycistronic Reporter System Reveals Functional Editing Characteristics of CRISPR-Cas.

The green fluorescent protein (GFP)-based reporter system has been widely harnessed as a quick quantitative activity assessment method for characterizing CRISPR-Cas via flow cytometry. However, due to the small size (738 nt) of the GFP coding sequence, the targeting sites for certain CRISPR-Cas are greatly restricted. To address this, here we developed a GFP tagged polycistronic reporter system to determine the activity of CRISPR-Cas in human cells. Specifically, the system contains the herpes simplex virus thymidine kinase (TK) gene, bacterial neomycin phosphotransferase (Neo) gene, and green fluorescent protein (GFP), named TNG gene, with a coding sequence of 2,577 nt. To investigate its performance, we generated a human cell line harboring the TNG expression cassette at the AAVS1 locus, and then we tested it with different Cas orthologs (SaCas9, St1Cas9, and AsCas12a). Our results demonstrated that using the TNG reporter system greatly expands the targeting site selection (3- to 13-fold) with CRISPR-Cas genome editing. The study therefore reports an additional method for the characterization of CRISPR-Cas technology.

An ultrasensitive, rapid and portable method for screening oseltamivir-resistant virus based on CRISPR/Cas12a combined with immunochromatographic strips.

Influenza is a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. The neuraminidase inhibitor oseltamivir is used as an antiviral drug in clinical treatment. However, its therapeutic effects can be greatly compromised by the emergence of drug-resistant mutant viruses. Thus, there is an urgent need to distinguish drug-resistant strains with a simple method. To address this, in the present study, we develop a rapid, sensitive and convenient molecular diagnosis method based on CRISPR/Cas12a technology and lateral flow detection (LFD). By targeting mutant sequences amplified by recombinase polymerase amplification (RPA) reaction, crRNA is designed to develop the CRISPR/Cas12a assay, and 2000 copies can be directly observed by the naked eye under blue light-emitting diode (LED) light. Combined with LFD, the limit of detection of RPA-CRISPR/Cas12a-LFD is about 20 copies of target sequence per reaction. Collectively, RPA-CRISPR/Cas12a-LFD method provides a novel alternative for the sensitive, specific and portable detection to diagnose oseltamivir-resistant mutant strains.

Novel Arginine End-Tagging Antimicrobial Peptides to Combat Multidrug-Resistant Bacteria.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them in vitro and in vivo. Our results demonstrate that Pep 6, one of the engineered peptides, shows great potential in combating Escherichia coli bacteremia and the Staphylococcus aureus skin burn infection model, which induces a 62-90% reduction in bacterial burden. Remarkably, after long serial passages of S. aureus and E. coli for 30 days, Pep 6 is still highly efficient in killing pathogens, compared with 64- and 128-fold increase in minimal inhibitory concentrations (MICs) for vancomycin and polymyxin B, respectively. We also found that Pep 6 exhibited robust biofilm-inhibiting activity and eliminated 61.33% of the mature methicillin-resistant Staphylococcus aureus (MRSA) biofilm with concentration in the MIC level. These results suggest that the RWWWR motif and binding of arginine end-tagging could be harnessed as a new agent for combating multidrug-resistant bacteria.

Genome editing of Corynebacterium glutamicum mediated with Cpf1 plus Ku/LigD.

OBJECTIVES: Corynebacterium glutamicum (C. glutamicum) has been harnessed for multi-million-ton scale production of glutamate and lysine. To further increase its amino acid production for fermentation industry, there is an acute need to develop next-generation genome manipulation tool for its metabolic engineering. All reported methods for genome editing triggered with CRISPR-Cas are based on the homologous recombination. While, it requires the generation of DNA repair template, which is a bottle-neck for its extensive application. RESULTS: In this study, we developed a method for gene knockout in C. glutamicum via CRISPR-Cpf1-coupled non-homologous end-joining (CC-NHEJ). Specifically, CRISPR-Cpf1 introduced double-strand breaks in the genome of C. glutamicum, which was further repaired by ectopically expressed two NHEJ key proteins (Mycobacterium tuberculosis Ku and ligase D). We provide the proof of concept, for CC-NHEJ, by the successful knockout of the crtYf/e gene in C. glutamicum with the efficiency of 22.00 ± 5.56%, or something like that. CONCLUSION: The present study reported a novel genome manipulation method for C. glutamicum.

FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella.

Eimeria species are intracellular parasites residing inside the intestinal epithelial cell, which cause poultry coccidiosis and result in significant financial losses in the poultry industry. Genome editing of Eimeria is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of Eimeria tenella (E. tenella). Ectopic expression of Cas9, i.e., via plasmids, would introduce transgene, which substantially limits its application, especially for vaccine development. In this study, we initially optimized the condition of the transfection protocol. We demonstrated that with the optimized condition, the transfection of FnCas12a (also known as "FnCpf1") protein and crRNA targeting EtHistone H4 triggered DNA double-strand breaks in vivo. We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase-thymidylate synthase gene (DHFR) as a selection marker to tag endogenous EtActin. The engineered E. tenella parasite possesses EYFP expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in E. tenella, which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for Eimeria species.

Panicle Apical Abortion 3 Controls Panicle Development and Seed Size in Rice.

BACKGROUND: In rice, panicle apical abortion is a common phenomenon that usually results in a decreased number of branches and grains per panicle, and consequently a reduced grain yield. A better understanding of the molecular mechanism of panicle abortion is thus critical for maintaining and increasing rice production. RESULTS: We reported a new rice mutant panicle apical abortion 3 (paa3), which exhibited severe abortion of spikelet development on the upper part of the branches as well as decreased grain size over the whole panicle. Using mapping-based clone, the PAA3 was characterized as the LOC_ Os04g56160 gene, encoding an H+-ATPase. The PAA3 was expressed highly in the stem and panicle, and its protein was localized in the plasma membrane. Our data further showed that PAA3 played an important role in maintaining normal panicle development by participating in the removal of reactive oxygen species (ROS) in rice. CONCLUSIONS: Our studies suggested that PAA3 might function to remove ROS, the accumulation of which leads to programmed cell death, and ultimately panicle apical abortion and decreased seed size in the paa3 panicle.

MULTI-FLORET SPIKELET 2, a MYB Transcription Factor, Determines Spikelet Meristem Fate and Floral Organ Identity in Rice.

An understanding of flower and panicle development is crucial for improving yield and quality in majority of grass crops. In this study, we used mapping-based cloning to identify MULTI-FLORET SPIKELET2 (MFS2), which encodes a MYB transcription factor and regulates flower and spikelet development in rice (Oryza sativa). In the mfs2 mutant, specification of palea identity was severely disturbed and showed degradation or transformation into a lemma-like organ, and the number of all floral organs was increased to varying degrees. Due to the increase in the number of floral organs and development of extra transformed palea/marginal region of the palea-like organs, some mfs2 spikelets had a tendency to produce two florets. These defects implied that the mfs2 mutation caused abnormal specification of palea identity and partial loss of spikelet determination. We confirm that MFS2 is a transcriptional repressor that shows strong repression activity by means of two typical ethylene-responsive element binding factor-associated amphiphilic motifs, one of which locates at the C terminus and is capable of interaction with three rice TOPLESS and TOPLESS-related proteins. The results indicate that MFS2 acts as a repressor that regulates floral organ identities and spikelet meristem determinacy in rice by forming a repression complex with rice TOPLESS and TOPLESS-related proteins.

High-fidelity SaCas9 identified by directional screening in human cells.

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.

Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells.

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) of bacterial adaptive immunity have been adopted as a powerful and versatile tool for manipulation of the genome. This paradigm has been widely applied in biological research and treatments of animal or cellular disease models. A critical feature of CRISPR-Cas is the protospacer adjacent motif (PAM), which dictates the DNA target recognition mechanism of Cas proteins. While, direct identifying functional PAM sequences in human cells remains a challenge. Here, we developed a positive screen system termed PAM-DOSE (PAM Definition by Observable Sequence Excision) to delineate the functional PAMs in human cells. Specifically, the PAM libraries for CRISPR-Cas (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a and MbCas12a) were generated and the corresponding CRISPR-Cas mediated cleaved fragments with functional PAM in human cells were harvested for DNA sequencing, which could be tracked and visualized with either florescence microscopy or flow cytometry analysis. With this system, we identified the functional PAMs of CRISPR-Cas members. We also found that spacer sequence affects the PAM preference of Cas proteins. This method will facilitate identification of functional PAMs for Cas-mediated human genome editing applications.

Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNAGln-processing system in human cells.

CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5' nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln-sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.

[Application and optimization of CRISPR/Cas system in bacteria].

Clustered regular interspaced short palindromic repeats (CRISPR) system has been widely used in recent years. Compared with traditional genome editing technology, CRISPR/Cas system has notable advantages, including high editing efficiency, high specificity, low cost and the convenience for manipulation. Type Ⅱ and Ⅴ CRISPR/Cas system only requires a single Cas9 protein or a single Cpf1 protein as effector nucleases for cutting double-stranded DNA, developed as genome editing tools. At present, CRISPR/Cas9 technology has been successfully applied to the genome editing of eukaryotes such as zebrafish, mice and human cells, whereas limited progress has been made in the genome editing of bacteria. In our review, we describe CRISPR/Cas system, its mechanism and summarize the optimization and progress of genome editing in bacteria.

Optimizing a CRISPR-Cpf1-based genome engineering system for Corynebacterium glutamicum.

BACKGROUND: Corynebacterium glutamicum is an important industrial strain for the production of a diverse range of chemicals. Cpf1 nucleases are highly specific and programmable, with efficiencies comparable to those of Cas9. Although the Francisella novicida (Fn) CRISPR-Cpf1 system has been adapted for genome editing in C. glutamicum, the editing efficiency is currently less than 15%, due to false positives caused by the poor targeting efficiency of the crRNA. RESULTS: To address this limitation, a screening strategy was developed in this study to systematically evaluate crRNA targeting efficiency in C. glutamicum. We quantitatively examined various parameters of the C. glutamicum CRISPR-Cpf1 system, including the protospacer adjacent motif (PAM) sequence, the length of the spacer sequence, and the type of repair template. We found that the most efficient C. glutamicum crRNA contained a 5'-NYTV-3' PAM and a 21 bp spacer sequence. Moreover, we observed that linear DNA could be used to repair double strand breaks. CONCLUSIONS: Here, we identified optimized PAM-related parameters for the CRISPR-Cpf1 system in C. glutamicum. Our study sheds light on the function of the FnCpf1 endonuclease and Cpf1-based genome editing. This optimized system, with higher editing efficiency, could be used to increase the production of bulk chemicals, such as isobutyrate, in C. glutamicum.

Engineering the Direct Repeat Sequence of crRNA for Optimization of FnCpf1-Mediated Genome Editing in Human Cells.

FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5'-KYTV-3'), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5'-TTTN-3' PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing. In principle, its engineering should influence the corresponding genome-editing activity and fidelity. Here we showed that the DR mutants [G(-9)A and U(-7)A] could modulate FnCpf1 performance in human cells, enabling enhancement of both genome-editing efficiency and fidelity. These newly identified features will facilitate the design and optimization of CRISPR-Cpf1-based genome-editing strategies.

A Single Multiplex crRNA Array for FnCpf1-Mediated Human Genome Editing.

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome.

CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways.

A novel mutation of p.F32I in GJA8 in human dominant congenital cataracts.

AIM: To identify a causative mutation in a three-generation family with autosomal dominant congenital total cataract and dissect the molecular consequence of the identified mutation. METHODS: Clinical and ophthalmological examinations were performed on the affected and unaffected family members. Mutation were screened in recruited family members by polymerase chain reaction (PCR) of the two reported genes (CRYAA and GJA8) which were linked to human total cataracts and direct sequencing of the PCR product. The molecular consequences of the identified mutation was dissected. The plasmids carrying wild-type and mutant mouse ORF of Gja8, coding for connexin 50 (Cx50), were generated and ectopic expressed in 293 cells. Recombinant protein expression and cellular localization of recombinated Cx50 were assessed by confocal microscopy. RESULTS: Clinical and ophthalmological examinations were performed on the affected and unaffected family members. Mutation were screened in recruited family members by PCR of the two reported genes (CRYAA and GJA8) which were linked to human total cataracts and direct sequencing of the PCR product. The molecular consequences of the identified mutation was dissected. The plasmids carrying wild-type and mutant mouse ORF of Gja8, coding for Cx50, were generated and ectopic expressed in 293 cells. Recombinant protein expression and cellular localization of recombinated Cx50 were assessed by confocal microscopy. CONCLUSION: This study has identified a novel cataract mutation in GJA8, which adds a novel mutation to the existing spectrum of Cx50 mutations with cataract. The molecular consequences of p.F32I mutation in GJA8 exclude instability and the mislocalization of mutant Cx50 protein.