Single-guide RNA Research Articles

Lipofection of Single Guide RNA Targeting MMP8 Decreases Proliferation and Migration in Lung Adenocarcinoma Cells.

Background and Objectives: Matrix metalloproteinases (MMP) have been implicated as major determinants of tumour growth and metastasis, which are considered two of the main hallmarks of cancer. The interaction of MMP8 and other signalling molecules within and adjacent tumoral tissues, including immune cells, are rather elusive, particularly of adenocarcinoma cell type. In this study, we aimed to investigate the role of MMP8 in non-small cell lung cancer proliferation and invasiveness potential. Materials and Methods: We individually lipofected with two different single guide RNA (sgRNAs) that specifically targeted on MMP8, with CRISPR-Cas 9 protein into the cells. Results: Our results clearly indicated that the lipofection of these complexes could lead to reduced ability of A549 cells to survive and proliferate to form colonies. In addition, when compared to non-transfected cells, the experimental cell groups receiving sgRNAs demonstrated relatively decreased migration rate, hence, wider wound gaps in scratch assay. The quantitative real time-polymerase chain reaction (qRT-PCR) demonstrated significant reduction in the MAP-K, survivin and PI3-K gene expression. MMP8 might have protective roles over tumour growth and spread in our body. Conclusions: The delivery of sgRNAs targeting on the MMP8 gene could induce tumour cell death and arrest cell migratory activity.

JMJD6 inhibits NF-κB Activation by Demethylating R149 of p65 Subunit to Attenuate Isoproterenol-Induced Cardiac Hypertrophy

Histone modification plays an important role in pathological cardiac hypertrophy and heart failure. However, the mechanisms underlying pathological cardiac hypertrophy still remain to be elucidated, especially involving histone arginine demethylation. Here, we investigated the roles of histone arginine demethylase jumonji domain-containing protein 6 (JMJD6) in isoproterenol (ISO)-induced pathological cardiac hypertrophy. JMJD6 level was upregulated in hypertrophic and ejection fraction preserved heart failure (EFpHF) rat cardiac tissues, while it was decreased in ejection fraction reduced heart failure (EFrHF) patients. JMJD6 overexpression attenuated ISO-induced cardiac hypertrophy as reflecting by the reduction of cardiomyocyte surface area and hypertrophic genes expression in cardiomyocytes. Meanwhile, cardiac-specific JMJD6 overexpression protected the hearts against ISO-induced cardiac hypertrophy, fibrosis, and rescued cardiac function. Conversely, depletion of JMJD6 by single-guide RNA (sgRNA) promoted ISO-induced hypertrophic responses in cardiomyocytes. We further demonstrated that JMJD6 interacts with nuclear factor kappa B (NF-κB) p65 in cytoplasm and reduces the nucleus level of p65 under hypertrophic stimulation in vivo and in vitro. Mechanistically, JMJD6 binds to p65 and demethylate p65 at arginine (R) 149 to inhibit the nuclear translocation of p65, and then inactivated NF-κB signaling, thereby protecting against pathological cardiac hypertrophy. We also found that JMJD6 demethylates histone H3R8, which may a new histone substrate of JMJD6. These findings indicate that JMJD6 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.

Abstract 2924: In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes

Abstract Microsatellite instability is a hallmark of DNA mismatch repair (MMR)-deficient colorectal cancer (CRC), and is accompanied by genome-wide somatic hypermutation and enhanced immunogenicity. Clinical trials have demonstrated remarkable efficacy of immune checkpoint blockade (i.e. anti-PD-1) in treating MMR-deficient cancers, which is thought to be driven by an enhanced burden of mutation-derived tumor specific antigens (neoantigens). Despite these successes, tumor mutation burden is an imperfect clinical predictor of immunotherapy response, and major questions remain regarding the mechanistic role of neoantigens and other features of MMR-deficiency in the anti-tumor immune response. Notably, most genetically engineered mouse models (GEMMs) fail to recapitulate the mutation burden of their associated human cancer and lack prominent immune involvement, precluding careful study of these questions. To model microsatellite instability in colon cancer, we developed novel autochthonous GEMMs utilizing CRISPR/Cas9-based knockout of DNA MMR genes in the distal colon. Colonoscopy-guided submucosal injection was used to deliver lentivirus containing two single-guide RNAs (sgRNAs) against Apc (the initiating event in the majority of colon cancers) and MMR pathway genes (Msh2, Mlh1, Msh3, Msh6). Whole-exome sequencing of MMR-targeted colon tumors revealed a high mutation burden with signatures consistent with those found in MMR-deficient human cancer. In addition, sequencing revealed a number of recurrently mutated microsatellites, including those within putative tumor suppressor genes Asxl1, Acvr2a, and Acvr1b. To interrogate the functional relevance of these genes in MMR-deficient colon cancer, we developed a novel ex vivo organoid competition assay. Specifically, Apc null organoids expressing Cas9 were infected with lentivirus expressing the fluorophore mScarlet and sgRNAs targeting the putative tumor suppressor genes. Flow cytometry was used to longitudinally assess kinetics of the mScarlet positive fraction. Preliminary results suggest a selective advantage following deletion of Asxl1 and Acvr2a. Further assessment of tumorigenesis in vivo will be performed to confirm the role of these putative tumor suppressors. Altogether, these genetically manipulable systems can be used for rapid functional interrogation of tumor suppressor genes, facilitating downstream mechanistic studies and preclinical testing of therapies in these important subsets of CRC. Citation Format: Daniel D. Zhang, Peter M. Westcott, Olivia Smith, Nate Sacks, Haley Hauck, Mary C. Beytagh, Abbey Jin, Tyler Jacks. In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2924.

English

Improved agricultural production of essential crops through advanced breeding is important for increasing access to nutritious food for the world's rapidly growing population, which is expected to reach 9.8 billion by 2050. Recent advancements in the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein9 (CRISPR/Cas9) genome editing process, which uses single-guide RNA for genome editing, have made it easy, stable and efficient tool for targeted gene mutations, knockout and knock-in/replacement to boost crop yield. The CRISPR/Cas method is constantly being improved, and its applications have greatly expanded. It can be used to modify the genome sequence of any organism, including plants like cassava, to achieve the desired trait. As a result, CRISPR/Cas is regarded as a game-changing technology in plant biology. Here, we discuss the principles of operation, implementations and future prospects of CRISPR/Cas9 for efficient processing of individual genes in cassava cultures. Recent work on cassava crop with regards to the use of CRISPR/Cas9 for the plant improvement was also addressed.   Key words:  Manihot esculenta, CRISPR/Cas9, genome editing, gRNA, protospacer adjacent motif (PAM).

Disruption of CTCF Boundary Remodels Chromatin Domain and Reprograms HOX Gene Transcription in Acute Myeloid Leukemia

The organization of the mammalian genome into separate domains either facilitates or restricts interactions between regulatory elements, e.g. enhancers and promoters, for normal gene regulation. CTCF insulator constrains the temporal HOX gene expression patterns within the confined chromatin domain for normal development. However, dysregulation of HOX genes is also a common feature of acute myeloid leukemia (AML). The molecular mechanisms of aberrant HOX expression and associated AML pathogenesis remain unclear. Here, we found that CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) acts as a chromatin boundary to establish and maintain AML specific aberrant HOX gene expression patterns and is essential for pathogenesis of myeloid malignancies.To unbiasedly and systematically investigate the mechanism underlying HOX gene regulation in AML, we employed a pooled CRISPR-Cas9 genetic knockout library screening to interrogate intergenic regulatory and chromatin boundary elements in all 4 HOX gene loci. Through this screening, we identified the CBS7/9 chromatin boundary located between HOXA7 and HOXA9 genes that is critical for maintaining aberrant expression of posterior HOXA genes including oncogene HOXA9 and HOXA10 in primary AML patients and cells. To further confirm the role of CBS7/9 in HOX gene regulation, two single-guide RNAs (sgRNAs) flanking 47 base pairs (bps) of the core CTCF motif located between HOXA7 and HOXA9 geneswere employed to guild Cas9 for deletion of the CBS7/9 boundary in 3 different subtypes of AML cells containing MLL rearrangements or NPM1C+ mutation. The defected CBS7/9 resulted in a decrease in CTCF binding to the caudal HOXA domain and an inhibition of posterior HOXA gene expression.To further delineate the role of CBS7/9 chromatin boundary in HOX gene organization and regulation, we then carried out genome wide ChIP-seq, ATAC-seq, 4C, and RNA-seq analysis to examine the changes in chromatin domain organization and corresponding gene expression patterns compared wild-type control and the CBS7/9 KO AML cells. Disruption of CBS7/9 boundary resulted in a spreading of repressive H3K27me3 domain into the caudal HOXA domain that subsequently impaired AML associated active TAD domain by altering enhancer/promoter chromatin accessibility and reducing ectopic interactions among the posterior HOXA genes. Consistent with the role of CBS7/9 boundary in HOX locus chromatin structure and organization, impairment of CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid specific transcriptome profiles important for pathogenesis of myeloid malignancies suggesting that the CBS7/9 boundary mediated ectopic HOX expression patterns might be essential for pathogenesis of myeloid malignancies. To further test this possibility, the CBS 7/9 boundary was deleted in both MLL rearranged AML cells and primary patient samples by the CRISPR-Cas9 mediated genome editing and the edited cells were transplanted into NSG mice for the development of human AML in mice. Disruption of CBS chromatin boundary in the HOXA locus reduced human leukemic blast development and invasion as well as enhanced survival of both transplanted AML cell xenograft and patient derived xenograft (PDX) mouse models. Thus, our finding demonstrated that CTCF boundary not only constrains normal gene expression program for memory of cell identity, but also plays a role in maintaining oncogenic transcription program for leukemic transformation. Alteration of CTCF mediated topological boundaries in the oncogene loci provides therapeutic approaches for targeting oncogenic expression program and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Targeting Mitochondrial Translation Overcomes Venetoclax Resistance in Acute Myeloid Leukemia (AML) through Activation of the Integrated Stress Response

Venetoclax is a highly specific BCL-2 inhibitor with promising activity against AML including leukemic stem cells (LSCs). However, venetoclax monotherapy in AML patients had limited clinical efficacy due to intrinsic or acquired drug resistance which has been attributed to the overexpression of other anti-apoptotic proteins including MCL-1 and BCL-xL. These findings highlight the importance of combination therapy to overcome resistance but the optimal strategy to achieving this goal is unclear.To address this issue, we generated a panel of highly resistant clones derived from the intrinsically sensitive MOLM-13 and MV-4-11 AML cell lines. Surprisingly, treatment with MCL-1 or BCL-xL inhibitors failed to overcome resistance despite higher expression of the anti-apoptotic proteins in resistant cells. To identify novel targets, we performed a genome-wide CRISPR knockout screen to find genes that upon inactivation, restore sensitivity to venetoclax. We transduced one of the resistant clones engineered to stably express Cas9 with a pooled lentiviral single-guide RNA (sgRNA) library that targets 17,661 protein-coding genes with 91,320 unique sgRNA sequences. Transduced cells were divided into 2 populations with one treated with venetoclax and the other untreated as control. The transduced cells were cultured for 29 days to negatively select sgRNAs that re-sensitized resistant cells to venetoclax. Cell samples were collected at regular intervals and subjected to next-generation sequencing of the sgRNA target region to quantify the abundance of each construct.We identified genes that were negatively selected in the presence of venetoclax using the MAGeCK algorithm. The top-ranked genes were highly enriched for gene ontology terms related to mitochondrial translation. We confirmed that RNAi-mediated knockdown of the top-ranked ribosomal subunit gene, DAP3, overcame venetoclax resistance. Pharmacologic inhibition of mitochondrial translation with a panel of protein synthesis inhibitor antibiotics similarly restored sensitivity in AML cell lines with intrinsic or acquired resistance. Tedizolid, a FDA-approved second generation oxazolidinone antibiotic, was the most effective in mediating this effect at clinically relevant concentrations. Furthermore, the combination of tedizolid and venetoclax targeted the LSC-enriched CD34+CD38- population in ex vivo -cultured primary AML samples. Importantly, normal cord blood hematopoietic stem and progenitor cells were more resistant to this combination suggestive of a wide therapeutic index .To decipher the mechanism by which tedizolid overcomes venetoclax resistance, we first profiled the expression of BCL-2 family members in total cellular and purified mitochondrial extracts. Antibiotic treatment did not affect total BCL-2, MCL-1, or BCL-xL protein levels but caused a substantial increase in BCL-2 levels in the mitochondrial fraction indicative of BCL-2 translocation to the organelle. Next, we confirmed that tedizolid treatment selectively reduced the expression of mitochondrial over nuclear-encoded proteins consistent with its inhibitory effect on mitochondrial translation. This mitonuclear protein imbalance has been shown to activate the integrated stress response (ISR). Tedizolid treatment activated this response as evidenced by an increase in eIF2-α phosphorylation and expression of ATF4, a master transcription factor regulator. To determine whether ISR activation is sufficient to overcome venetoclax resistance, we treated resistant cells with a small-molecule activator of PERK (CCT020312), which activates the ISR through direct eIF2-α phosphorylation, and observed re-sensitization to venetoclax.In summary, our results demonstrate that inhibition of mitochondrial translation, which activates ISR and triggers BCL-2 translocation to the mitochondria, is a novel approach to overcoming venetoclax resistance in AML cells. Our findings provide the rationale for exploring the use protein synthesis inhibitor antibiotics or direct ISR activators in combination with venetoclax for the treatment of AML or other malignancies. DisclosuresNo relevant conflicts of interest to declare.

Gene editing in a Myo6 semi-dominant mouse model rescues auditory function

Myosin VI（MYO6） is an unconventional myosin that is vital for auditory and vestibular function. Pathogenic variants in the human MYO6 gene cause autosomal-dominant or -recessive forms of hearing loss. Effective treatments for Myo6 mutation causing hearing loss are limited. We studied whether adeno-associated virus (AAV)-PHP.eB vector-mediated invivo delivery of Staphylococcus aureus Cas9 (SaCas9-KKH)-single-guide RNA (sgRNA) complexes could ameliorate hearing loss in a Myo6WT/C442Y mouse model that recapitulated the phenotypes of human patients. The invivo editing efficiency of the AAV-SaCas9-KKH-Myo6-g2 system on Myo6C442Y is 4.05% on average in Myo6WT/C442Y mice, which was ∼17-fold greater than editing efficiency of Myo6WT alleles. Rescue of auditory function was observed up to 5months post AAV-SaCas9-KKH-Myo6-g2 injection in Myo6WT/C442Y mice. Meanwhile, shorter latencies of auditory brainstem response (ABR) wave I, lower distortion product otoacoustic emission (DPOAE) thresholds, increased cell survival rates, more regular hair bundle morphology, and recovery of inward calcium levels were also observed in the AAV-SaCas9-KKH-Myo6-g2-treated ears compared to untreated ears. These findings provide further reference for invivo genome editing as a therapeutic treatment for various semi-dominant forms of hearing loss and other semi-dominant diseases.

Control of Plant Viral Diseases by CRISPR/Cas9: Resistance Mechanisms, Strategies and Challenges in Food Crops.

Protecting food crops from viral pathogens is a significant challenge for agriculture. An integral approach to genome-editing, known as CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9), is used to produce virus-resistant cultivars. The CRISPR/Cas9 tool is an essential part of modern plant breeding due to its attractive features. Advances in plant breeding programs due to the incorporation of Cas9 have enabled the development of cultivars with heritable resistance to plant viruses. The resistance to viral DNA and RNA is generally provided using the Cas9 endonuclease and sgRNAs (single-guide RNAs) complex, targeting particular virus and host plant genomes by interrupting the viral cleavage or altering the plant host genome, thus reducing the replication ability of the virus. In this review, the CRISPR/Cas9 system and its application to staple food crops resistance against several destructive plant viruses are briefly described. We outline the key findings of recent Cas9 applications, including enhanced virus resistance, genetic mechanisms, research strategies, and challenges in economically important and globally cultivated food crop species. The research outcome of this emerging molecular technology can extend the development of agriculture and food security. We also describe the information gaps and address the unanswered concerns relating to plant viral resistance mediated by CRISPR/Cas9.

All-In-One Dendrimer-Based Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via homology-directed repair (HDR) that requires careful orchestration of complex steps. Herein, dendrimer-based lipid nanoparticles (dLNPs) are engineered to co-encapsulate and deliver multiple components for in vivo HDR correction. BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation are employed to assess HDR-mediated gene editing following simultaneous, one-pot delivery of Cas9 mRNA, single-guide RNA, and donor DNA. Molar ratios of individual LNP components and weight ratios of the three nucleic acids are systematically optimized to increase HDR efficiency. Using flow cytometry, fluorescence imaging, and DNA sequencing to quantify editing, optimized 4A3-SC8 dLNPs edit >91%of all cells with 56% HDR efficiency in vitro and >20%HDR efficiency in xenograft tumors in vivo. Due to the all-in-one simplicity and high efficacy, the developed dLNPs offer a promising route toward the gene correction of disease-causing mutations.

Genome- and transcriptome-wide off-target analyses of an improved cytosine base editor.

Cytosine base editors (CBEs) are the promising tools for precise genome editing in plants. It is important to investigate potential off-target effects of an efficient CBE at the genome and transcriptome levels in a major crop. Based on comparison of five cytidine deaminases and two different promoters for expressing single-guide RNAs (sgRNAs), we tested a highly efficient A3A/Y130F-BE3 system for efficient C-to-T base editing in tomato (Solanum lycopersicum). We then conducted whole-genome sequencing of four base-edited tomato plants, three Green fluorescent protein (GFP)-expressing control plants, and two wild-type plants. The sequencing depths ranged from 25× to 49× with read mapping rates >97%. No sgRNA-dependent off-target mutations were detected. Our data show an average of approximately 1,000 single-nucleotide variations (SNVs) and approximately 100 insertions and deletions (indels) per GFP control plant. Base-edited plants had on average elevated levels of SNVs (approximately 1,250) and indels (approximately 300) per plant. On average, about 200 more C-to-T (G-to-A) mutations were found in a base-edited plant than a GFP control plant, suggesting some level of sgRNA-independent off-target effects, though the difference is not statistically significant. We also conducted RNA sequencing of the same four base-edited plants and three GFP control plants. An average of approximately 200 RNA SNVs was discovered per plant for either base-edited or GFP control plants. Furthermore, no specific enrichment of C-to-U mutations can be found in the base-edited plants. Hence, we cannot find any evidence for bona fide off-target mutations by A3A/Y130F-BE3 at the transcriptome level.

RNA Binding Proteins As Regulators of Oxidative Stress Identified by a Targeted CRISPR-Cas9 Single Guide RNA Library.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing system has been broadly adopted for high-throughput genetic screens. However, the application of genome-wide single guide RNA (sgRNA) libraries can be challenging. We generated a custom sgRNA library, an order of magnitude smaller than genome-wide alternatives, to facilitate the genetic screening of RNA binding proteins (RBPs). We demonstrated the utility of our reagent in a genetic screen for RBPs that conveyed cellular resistance or sensitivity to oxidative stress induced by paraquat. This identified that CSDE1 and STRAP, proteins that interact with each other, convey sensitivity to oxidative stress and that Pumilio homologues (PUM1 and PUM2) convey resistance. Targeting eIF4-E1 and -A1 protected cells from high-dose paraquat, whereas eIF4E2 targeted cells did less well. We also found that G3BP1 promoted sensitivity to a low dose of paraquat but protected cells at a higher dose. Our study highlights the use of genetic screens to identify roles of RBPs and identifies novel genes regulating sensitivity to oxidative stress.

Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors.

Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.

Deep Sequencing Reveals the Comprehensive CRISPR-Cas9 Editing Spectrum in Bombyx mori.

Application of the clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) technology has revolutionized biology by greatly enhancing the ability to introduce mutations into DNA for research and prospective therapeutic purposes. However, the understanding of Cas9 editing outcomes is still limited. Previously, it was considered that Cas9 introduces stochastic insertions or deletions (indels) at the target site. In the current study, we performed in vivo multiplex editing, deep sequencing, and comprehensive analysis of its editing outcomes in Bombyx mori (B. mori). A total of 31161 editing events from 9 single-guide RNA (sgRNA) sites in 16 individuals were generated and analyzed, and we found that Cas9 introduces mutations with some regularity rather than via stochastic indels. The editing efficiency varies with sgRNA sequences, individuals, and orientation. Small deletions account for the vast majority of mutated sequences, followed by a small fraction of substitutions and insertions. The most likely mutations are deletions between two microhomologous sequences or single-base deletions at the cleavage site in the absence of microhomologous pairs. Insertions are formed by diverse mechanisms, including direct acquisition of free genomic fragments, duplication of broken ends, replication of adjacent sequences, or random addition of free nucleotides. The above results indicate that the Cas9 editing spectrum is reproducible and predictable. Thus, our findings enable a deeper understanding of Cas9-mediated mutagenesis and better design of genome editing experiments, as well as elucidate the DNA double-strand break repair processes in B. mori.

In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels

Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

SgRNA-RF: Identification of SgRNA On-Target Activity With Imbalanced Datasets.

Single-guide RNA is a guide RNA (gRNA), which guides the insertion or deletion of uridine residues into kinetoplastid during RNA editing. It is a small non-coding RNA that can be combined with pre -mRNA pairing. SgRNA is a critical component of the CRISPR/Cas9 gene knockout system and play an important role in gene editing and gene regulation. It is important to accurately and quickly identify highly on-target activity sgRNAs. Due to its importance, several computational predictors have been proposed to predict sgRNAs on-target activity. All these methods have clearly contributed to the development of this very important field. However, they also have certain limitations. In the paper, we developed a new classifier SgRNA-RF, which extracts the features of nucleic acid composition and structure of on-target activity sgRNA sequence and identified by random forest algorithm. In addition to solving an imbalanced dataset, this paper proposed a new method called CS-Smote. We compared sgRNA-RF with state-of-the-art predictors on the five datasets, and found SgRNA-RF significantly improved the identification accuracy, with accuracies of 0.8636,0.9161,0.894,0.938,0.965,0.77,0.979,0.973, respectively. The user-friendly web server that implements sgRNA-RF is freely available at http://server.malab.cn/sgRNA-RF/.

Scalable recombinase-based gene expression cascades

Temporal modulation of the expression of multiple genes underlies complex complex biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. Here, we describe a modular recombinase-based gene circuit architecture, comprising tandem gene perturbation cassettes (GPCs), that enables the sequential expression of multiple genes in a defined temporal order by alternating treatment with just two orthogonal ligands. We use tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades that trigger the sequential accumulation of mutations. We build an all-in-one gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within a gene expression cascade, and deletes itself for safety. Tandem GPCs offer a multi-tiered cellular programming tool for modeling multi-stage genetic changes, such as tumorigenesis and cellular differentiation.

Prospective gene therapy design against coronavirus COVID-19 by recruiting CRISPR Cas9 approach

A novel Coronavirus disease (COVID-19) has being originated from animals and causing to running outbreak of viral pneumonia in human all over the world. The World Health Organization (WHO) has considered COVID-19 infection as Epidemic disease in March 2020. The high percentages of death rate among people lead the researchers and scientists in different fields of medicine in order to find solution for this threating problem. In this regard, an editing gene technique was employed in this study, in order to deter the viability of coronavirus genetically. The main objective in this paper first has been determined and obtained the essential proteins of COVID-19 coronavirus proliferation by using NCBI website which included S, E, N, M, ORF3a, ORF6, ORF7a, ORF7b, ORF8, ORF10 proteins. The second objective is to use a very precise technique of editing gene called CRISPR-Cas9 to make changes to the virus’s RNA, through designing single-guide RNA for the essential each protein of COVID-19, in order to inactivate an effective certain protein. These techniques will be provided for each patient and healthy person by injection of all genes components using the Gene-gun machine or spray aerosol to make ensure reach it to the target cell.

Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA.

Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5' end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing.IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

Optimizing glyphosate tolerance in rapeseed by CRISPR/Cas9-based geminiviral donor DNA replicon system with Csy4-based single-guide RNA processing.

Rapeseed (Brassica napus L.) is an important oil crop worldwide, and effective weed control can protect its yield and quality. Farmers can benefit from cultivars tolerant to herbicides such as glyphosate. Amino acid substitutions in enolpyruvylshikimate-3-phosphate synthase (EPSPS) render the plant less sensitive to glyphosate. Therefore, we aimed to optimize the glyphosate tolerance trait in rapeseed via endogenous EPSPS modification. To achieve effective gene replacement in B. napus L., we employed a CRISPR/Cas9 system expressing single-guide RNAs (sgRNAs) cleaved by the CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa, for targeted induction of double-strand breaks. Both the donor template and a geminiviral replicon harbouring an sgRNA expression cassette were introduced into plant cells. Using sgRNAs targeting adjacent donor DNA template containing synonymous mutations in sgRNA sites, we achieved precise gene replacements in the endogenous B. napus EPSPS gene, BnaC04EPSPS, resulting in amino acid substitutions at frequencies up to 20%. Rapeseed seedlings harbouring these substitutions were glyphosate-tolerant. Furthermore, modifications in BnaC04EPSPS were precisely transmitted to the next generation. Our genome editing strategy enables highly efficient gene targeting and the induction of glyphosate tolerance in oilseed rape.

CHK2 Inhibition Provides a Strategy to Suppress Hematologic Toxicity from PARP Inhibitors.

Patients with cancer treated with PARP inhibitors (PARPi) experience various side effects, with hematologic toxicity being most common. Short-term treatment of mice with olaparib resulted in depletion of reticulocytes, B-cell progenitors, and immature thymocytes, whereas longer treatment induced broader myelosuppression. We performed a CRISPR/Cas9 screen that targeted DNA repair genes in Eμ-Myc pre-B lymphoma cell lines as a way to identify strategies to suppress hematologic toxicity from PARPi. The screen revealed that single-guide RNAs targeting the serine/threonine kinase checkpoint kinase 2 (CHK2) were enriched following olaparib treatment. Genetic or pharmacologic inhibition of CHK2-blunted PARPi response in lymphoid and myeloid cell lines, and in primary murine pre-B/pro-B cells. Using a Cas9 base editor, we found that blocking CHK2-mediated phosphorylation of p53 also impaired olaparib response. Our results identify the p53 pathway as a major determinant of the acute response to PARPi in normal blood cells and demonstrate that targeting CHK2 can short circuit this response. Cotreatment with a CHK2 inhibitor did not antagonize olaparib response in ovarian cancer cell lines. Selective inhibition of CHK2 may spare blood cells from the toxic influence of PARPi and broaden the utility of these drugs. IMPLICATIONS: We reveal that genetic or pharmacologic inhibition of CHK2 may offer a way to alleviate the toxic influence of PARPi in the hematologic system.