gRNA Sequences Research Articles

Abstract A017: IGF2BP1 is essential for ETO2/GLIS2 leukemogenesis via stabilization of primitive myeloid transcriptional programs

Abstract Certain subtypes of pediatric leukemia continue to have dismal cure rates despite advances in targeted therapy. One such disease is acute megakaryoblastic leukemia (AMKL) with a rearrangement of ETO2/GLIS2 (also known as CBFA2T3/GLIS2.) Based on our preliminary data utilizing CRISPR/Cas9 screens in vivo in patient-derived xenografts, we have nominated IGF2BP1 as a potential vulnerability in ETO2/GLIS2-rearranged AMKL. For our discovery phase, we utilized a patient-derived E2G2r AMKL which successfully engrafts in NSGS mice. We created a stable Cas9-expressing subclone using lentiviral transduction followed by FACS enrichment and limiting dilution transplants. We then generated a custom library targeting the top 100 overexpressed genes between E2G2r leukemias versus other leukemia PDXs which we previously characterized, including eight transcription factors with motifs enriched in differentially expressed genes, as well as eight pan-essential positive controls. Mice were sacrificed at humane endpoints, and spleens and bone marrow underwent PCR amplification for gRNA sequences followed by sequencing and analysis with MAGeCK-VISPR (Wei L, et al. Genome Biol 2015). We observed the dropout of 38 genes with a false discovery rate < 0.25, including all positive control gRNAs. Among the most significantly depleted genes was the RNA binding protein IGF2BP1. We subsequently characterized phenotypic changes using shRNAs in the E2G2r cell line M07e and ETO2/GLIS2-transformed CD34+ umbilical cord blood cells. Notably, we observed impaired cell growth with IGF2BP1 knockdown in both cell lines as well as the parent xenograft. We also generated M07e lines expressing a degron (FKBPF36V, aka “dTAG”, Nabet et al. Nat Chem Biol 2018) knocked in at the endogenous IGF2BP1 locus and observed similar phenomena. Through flow cytometry of M07e following IGF2BP1 depletion, we observed changes in the leukemic immunophenotype as well, suggesting perturbation of megakaryocyte differentiation potential. To determine the mechanism underlying our observations, we performed enhanced UV crosslinking with IP and sequencing (Blue SM et al. Nat Protoc 2022) against both IGF2BP1 and m6A residues based on the putative role of IGF2BP1 as an m6A reader. We observed binding of IGF2BP1 predominantly in 3’UTRs, as well as the presence of m6A-modified bases at IGF2BP1 binding sites. Gene ontology (GO) analysis of IGF2BP1-bound transcripts demonstrated enrichment for GO terms pertaining to embryonic hematopoiesis and megakaryocyte maturation/development. We further evaluated global changes in gene expression following IGF2BP1 loss, and performed RNAseq 72 hours following shRNA-mediated knockdown. GO analysis showed that downregulated genes were highly enriched for E2F-mediated transcriptional programs. We hypothesize that IGF2BP1 regulates myeloid developmental transcripts essential for E2G2r leukemogenesis, likely via stabilization in an m6A-dependent fashion, and thus may represent a selective therapeutic target. Work is ongoing to further elucidate this proposed mechanism. Citation Format: Jason Clark, Mark Wunderlich, Jing Chen, Daniel Starczynowski, Lynn Lee. IGF2BP1 is essential for ETO2/GLIS2 leukemogenesis via stabilization of primitive myeloid transcriptional programs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A017.

CRISPR-Cas9 guided RNA-based model for the silencing of spinal bulbar muscular atrophy: A functional genetic disorder.

This study explores a novel therapeutic approach for spinal bulbar muscular atrophy (SBMA), a neurodegenerative disorder caused by a mutation in the Androgen Receptor (AR) gene. The aim is to investigate the potential of CRISPR-Cas9 technology in targeting the mutant AR gene to inhibit its production. The objectives include assessing the accuracy and efficacy of CRISPR-Cas9 guided RNAs in silencing the mutant gene and evaluating the feasibility of this approach as a treatment for SBMA. Computational and in-silico approaches are used to evaluate the feasibility of using CRISPR-Cas9 technology for treating SBMA. Computational analysis is used to design CRISPR-Cas9 guided RNAs targeting the mutant AR gene, assessing their on-target and off-target scores, GC content, and structural accuracy. In-silico simulations predict the potential therapeutic outcomes of the CRISPR-Cas9 approach in an artificial environment. Three guided RNA (gRNA) sequences were designed using the CHOPCHOP tool, targeting specific regions of the AR gene with high efficiency and 100% match. These gRNAs demonstrated effective targeting with minimal off-target scores and optimal GC content. Additionally, lentiCRISPR v2 plasmids were designed for the delivery of CRISPR materials, enabling high-efficiency multiplex genome editing of the AR gene. Thermodynamic ensemble predictions indicated favorable secondary structure stability of the designed gRNAs, further supporting their suitability for gene editing. The evaluation of designed gRNAs confirmed their strong binding ability to the target sequences, validating their potential as effective tools for genome editing. The study highlights the potential of CRISPR-Cas9 technology for targeting the Androgen Receptor gene associated with spinal bulbar muscular atrophy (SBMA). The findings support the feasibility of this approach for gene editing and suggest further exploration in preclinical and clinical settings. Recommendations include continued research to optimize CRISPR-Cas9 delivery methods and enhance specificity for therapeutic applications in SBMA.

IgRNA Prediction and Selection AI Models (igRNA-PS) for Bystander-less ABE Base Editing

CRISPR derived base editing techniques tend to edit multiple bases in the targeted region, which impedes precise reversion of disease-associated single nucleotide variations (SNVs). We designed an imperfect gRNA (igRNA) editing strategy to achieve bystander-less single-base editing. To predict the performance and provide ready-to-use igRNAs, we employed a high-throughput method to edit 5000 loci, each with approximate 19 systematically designed ABE igRNAs. Through deep learning of the relationship of editing efficiency, original gRNA sequence and igRNA sequence, AI models were constructed and tested, designated igRNA Prediction and Selection AI models (igRNA-PS). The models have three functions, First, they can identify the major editing site from the bystanders on a gRNA protospacer with a near 90% accuracy. second, a modified single-base editing efficiency (SBE), considering both single-base editing efficiency and product purity, can be predicted for any given igRNAs. Third, for an editing locus, a set of 64 igRNAs derived from a gRNA can be generated, evaluated through igRNA-PS to select for the best performer, and provided to the user. In this work, we overcome one of the most significant obstacles of base editors, and provide a convenient and efficient approach for single-base bystander-less ABE base editing.

Graded-CRISPRi, a Tool for Tuning the Strengths of CRISPRi-Mediated Knockdowns in Vibrio natriegens Using gRNA Libraries.

In recent years, the fast-growing bacterium Vibrio natriegens has gained increasing attention as it has the potential to become a next-generation chassis for synthetic biology. A wide range of genetic parts and genome engineering methods have already been developed. However, there is still a need for a well-characterized tool to effectively and gradually reduce the expression levels of native genes. To bridge this gap, we created graded-CRISPRi, a system utilizing gRNA variants that lead to varying levels of repression strength. By incorporating multiple gRNA sequences into our design, we successfully extended this concept to simultaneously repress four distinct reporter genes. Furthermore, we demonstrated the capability of using graded-CRISPRi to target native genes, thereby examining the effect of various knockdown levels on growth.

A model for accurate quantification of CRISPR effects in pooled FACS screens.

CRISPR screens are powerful tools to identify key genes that underlie biological processes. One important type of screen uses fluorescence activated cell sorting (FACS) to sort perturbed cells into bins based on the expression level of marker genes, followed by guide RNA (gRNA) sequencing. Analysis of these data presents several statistical challenges due to multiple factors including the discrete nature of the bins and typically small numbers of replicate experiments. To address these challenges, we developed a robust and powerful Bayesian random effects model and software package called Waterbear. Furthermore, we used Waterbear to explore how various experimental design parameters affect statistical power to establish principled guidelines for future screens. Finally, we experimentally validated our experimental design model findings that, when using Waterbear for analysis, high power is maintained even at low cell coverage and a high multiplicity of infection. We anticipate that Waterbear will be of broad utility for analyzing FACS-based CRISPR screens.

Inhibiting renal Traf2 and NCK interacting kinase (TNIK) decreases NKKC2 phosphorylation and blunts salt-sensitive hypertension

The Na/K/2Cl cotransporter NKCC2 mediates the NaCl reabsorption in the thick ascending limb (TAL). Phosphorylation at Thr-96,101 and traffcking to the surface are increased in the Dahl salt-sensitive (DSS) rat together with SPAK phosphorylation, an upstream kinase for Thr-96,101 in NKCC2. We previously found that deletion of SPAK in the DSS, lowers NKCC2 phosphorylation and blunts salt-sensitive hypertension, but doesn’t completely abolish NKCC2 phosphorylation. Therefore, other kinases may be involved in NKCC2 phosphorylation. We recently found the Traf2 and NCK interacting kinase (TNIK) phosphorylates NKCC2 and is overexpressed in the TALs of DSS rats. We hypothesized that TNIK contributes to salt-sensitive hypertension by increasing NKCC2 hyperphosphorylation in DSS rats. We approached TNIK inhibition in two ways, using a pharmacological inhibitor and its genomic deletion with CRISPR/Cas9. First, we studied the small molecule TNIK inhibitor NCB-0846, and whether it could decrease baseline NKCC2 phosphorylation in TALs from DSS rats. Treating TALs from DSS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr-96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.001, n=3). To study in vivo effcacy of NCB-0846, we directly infused NCB (or vehicle) into the left kidney via implanted renal medullary catheters connected to minipumps in DSS rats fed 4% NaCl diet. We found that NCB-0846 decreased SBP by 30 mmHg within four days compared to the vehicle-infused group, and BP remained lower for 7 days (n=5, p<0.03 vs control). To decrease TNIK gene expression we used Cas9 mediated gene editing. For this, we designed gRNA sequences targeting TNIK and tested their effcacy in C6 cells. One of the gRNAs designed decreased TNIK expression by 48 ± 22% compared to control (p=0.101, n=3). To decrease TNIK in TALs in vivo, we injected the renal medulla of the left kidney with AAV-gRNA-TNIK together with AAV-NKCC2 promoter-Cas9. After 2 weeks, we isolated medullary TALs and measured TNIK expression and NKCC2 phosphorylation. TNIK expression was decreased by 50 ± 5.5% (n=4, p<0.0001), and pThr-96,101-NKCC2 was decreased by 38.1 ± 10.6% in transduced TALs compared to kidneys transduced with AV-control (n=4, p<0.05). The expression of related kinases SPAK and OSR1 was not modified (SPAK 79.2 ± 18.3%, OSR1 81.3 ± 13.3%, n=4, ns). We conclude that NKCC2 phosphorylation in the DSS rat is in part caused by TNIK and that this new kinase could be a target for salt sensitive hypertension. Future experiments will test the effect of gene editing TNIK in salt sensitive hypertension. 23POST1019882 AHA15GRNT25710369. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Molecular identification of Proteus mirabilis, Vibrio species leading to CRISPR-Cas9 modification of tcpA and UreC genes causing cholera and UTI

Heavy metal accumulation increases rapidly in the environment due to anthropogenic activities and industrialization. The leather and surgical industry produces many contaminants containing heavy metals. Cadmium, a prominent contaminant, is linked to severe health risks, notably kidney and liver damage, especially among individuals exposed to contaminated wastewater. This study aims to leverage the natural cadmium resistance mechanisms in bacteria for bioaccumulation purposes. The industrial wastewater samples, characterized by an alarming cadmium concentration of 29.6 ppm, 52 ppm, and 76.4 ppm—far exceeding the recommended limit of 0.003 ppm—were subjected to screening for cadmium-resistant bacteria using cadmium-supplemented media with CdCl2. 16S rRNA characterization identified Vibrio cholerae and Proteus mirabilis as cadmium-resistant bacteria in the collected samples. Subsequently, the cadmium resistance-associated cadA gene was successfully amplified in Vibrio species and Proteus mirabilis, revealing a product size of 623 bp. Further analysis of the identified bacteria included the examination of virulent genes, specifically the tcpA gene (472 bp) associated with cholera and the UreC gene (317 bp) linked to urinary tract infections. To enhance the bioaccumulation of cadmium, the study proposes the potential suppression of virulent gene expression through in-silico gene-editing tools such as CRISPR-Cas9. A total of 27 gRNAs were generated for UreC, with five selected for expression. Similarly, 42 gRNA sequences were generated for tcpA, with eight chosen for expression analysis. The selected gRNAs were integrated into the lentiCRISPR v2 expression vector. This strategic approach aims to facilitate precise gene editing of disease-causing genes (tcpA and UreC) within the bacterial genome. In conclusion, this study underscores the potential utility of Vibrio species and Proteus mirabilis as effective candidates for the removal of cadmium from industrial wastewater, offering insights for future environmental remediation strategies.

Prediction of base editor off-targets by deep learning

Due to the tolerance of mismatches between gRNA and targeting sequence, base editors frequently induce unwanted Cas9-dependent off-target mutations. Here, to develop models to predict such off-targets, we design gRNA-off- target pairs for adenine base editors (ABEs) and cytosine base editors (CBEs) and stably integrate them into the human cells. After five days of editing, we obtain valid efficiency datasets of 54,663 and 55,727 off-targets for ABEs and CBEs, respectively. We use the datasets to train deep learning models, resulting in ABEdeepoff and CBEdeepoff, which can predict off-target sites. We use these tools to predict off-targets for a panel of endogenous loci and achieve Spearman correlation values varying from 0.710 to 0.859. Finally, we develop an integrated tool that is freely accessible via an online web server http://www.deephf.com/#/bedeep/bedeepoff. These tools could facilitate minimizing the off-target effects of base editing.

Cytosine base editors optimized for genome editing in potato protoplasts.

In this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (Solanum tuberosum), which conferred up to 43% C-to-T conversion of all alleles in the protoplast pool. Earlier, gene-edited potato plants were successfully generated by polyethylene glycol-mediated CRISPR/Cas9 transformation of protoplasts followed by explant regeneration. In one study, a 3-4-fold increase in editing efficiency was obtained by replacing the standard Arabidopsis thaliana AtU6-1 promotor with endogenous potato StU6 promotors driving the expression of the gRNA. Here, we used this optimized construct (SpCas9/StU6-1::gRNA1, target gRNA sequence GGTC4C5TTGGAGC12AAAAC17TGG) for the generation of CBEs tailor-made for potato and tested for C-to-T base editing in the granule-bound starch synthase 1 gene in the cultivar Desiree. First, the Streptococcus pyogenes Cas9 was converted into a (D10A) nickase (nCas9). Next, one of three cytosine deaminases from human hAPOBEC3A (A3A), rat (evo_rAPOBEC1) (rA1), or sea lamprey (evo_PmCDA1) (CDA1) was C-terminally fused to nCas9 and a uracil-DNA glycosylase inhibitor, with each module interspaced with flexible linkers. The CBEs were overall highly efficient, with A3A having the best overall base editing activity, with an average 34.5%, 34.5%, and 27% C-to-T conversion at C4, C5, and C12, respectively, whereas CDA1 showed an average base editing activity of 34.5%, 34%, and 14.25% C-to-T conversion at C4, C5, and C12, respectively. rA1 exhibited an average base editing activity of 18.75% and 19% at C4 and C5 and was the only base editor to show no C-to-T conversion at C12.

GRNA-SeqRET: a universal tool for targeted and genome-scale gRNA design and sequence extraction for prokaryotes and eukaryotes.

High-throughput genetic screening is frequently employed to rapidly associate gene with phenotype and establish sequence-function relationships. With the advent of CRISPR technology, and the ability to functionally interrogate previously genetically recalcitrant organisms, non-model organisms can be investigated using pooled guide RNA (gRNA) libraries and sequencing-based assays to quantitatively assess fitness of every targeted locus in parallel. To aid the construction of pooled gRNA assemblies, we have developed an in silico design workflow for gRNA selection using the gRNA Sequence Region Extraction Tool (gRNA-SeqRET). Built upon the previously developed CCTop, gRNA-SeqRET enables automated, scalable design of gRNA libraries that target user-specified regions or whole genomes of any prokaryote or eukaryote. Additionally, gRNA-SeqRET automates the bulk extraction of any regions of sequence relative to genes or other features, aiding in the design of homology arms for insertion or deletion constructs. We also assess in silico the application of a designed gRNA library to other closely related genomes and demonstrate that for very closely related organisms Average Nucleotide Identity (ANI) > 95% a large fraction of the library may be of relevance. The gRNA-SeqRET web application pipeline can be accessed at https://grna.jgi.doe.gov. The source code is comprised of freely available software tools and customized Python scripts, and is available at https://bitbucket.org/berkeleylab/grnadesigner/src/master/ under a modified BSD open-source license (https://bitbucket.org/berkeleylab/grnadesigner).

CRISPR-Cas Systems: Programmable Nuclease Revolutionizing the Molecular Diagnosis.

CRISPR-Cas system has evolved as a highly preferred genetic engineering tool to perform target gene manipulation via alteration of the guide RNA (gRNA) sequence. The ability to recognize and cleave a specific target with high precision has led to its applicability in multiple frontiers pertaining to human health and medicine. From basic research focused on understanding the molecular basis of disease to translational approach leading to early and precise disease diagnosis as well as developing effective therapeutics, the CRISPR-Cas system has proved to be a quite versatile tool. The coupling of CRISPR-Cas mediated cleavage with isothermal amplification (ISA) of target DNA, followed by a read-out using fluorescent or colorimetric reporters appears quite promising in providing a solution to the urgent need for nucleic acid-based point-of-care diagnostic. Hence, it has been recognized as a highly sophisticated molecular diagnostic tool for the detection of disease-specific biomarkers not limited to nucleic acids-based detection but also of non-nucleic acid targets such as proteins, exosomes, and other small molecules. In this review, we have presented salient features and principles of class 2 type II, V, and VI CRISPR-Cas systems represented by Cas9, Cas12, and Cas13 endonucleases which are frequently used in molecular diagnosis. The article then highlights different medical diagnostic applications of CRISPR-Cas systems focusing on the diagnosis of SARS-CoV-2, Dengue, Mycobacterium tuberculosis, and Listeria monocytogenes. Lastly, we discuss existing obstacles and potential future pathways concerning this subject in a concise manner.

Abstract 250: Identifying drivers of liver cancer using CRISPR activation screening in vivo

Abstract Hepatocellular Carcinoma (HCC) is the most prevalent type of liver cancer and the third leading cause of cancer deaths worldwide. Drugs approved for first- and second-line therapy extend survival by only several months. Hence, there is still a pressing need for new and effective treatments. Sequencing technologies applied to human HCC tissues have identified hundreds of genes that undergo amplification and that may be correlated with mortality. Nevertheless, correlation does not equate to functionality. We aim to annotate gene function by employing an innovative approach with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and catalytically dead version of CRISPR-associated endonuclease (dCas9) in a CRISPR/dCas9 activation (CRISPRa) screen to identify driver genes of HCC tumorigenesis in mice. We deliver to the liver’s hepatocytes a gRNA library targeting genes of interest on plasmids that contain: (1) a transposon system for stable integration of DNA sequences into the cell genome, (2) Myc to drive hyperproliferation, as MYC is frequently over-expressed in human HCC, (3) synergistic activation mediator complex sequences for transcriptional activation of target genes, and (4) the gRNA sequence, the only variable sequence in the library. Using datasets from the Cancer Genome Atlas, we identified 51 genes that are both enriched and amplified in HCC patients. We included gRNAs targeting Tert, Vegfa, and Ccnd1, as control genes with known roles in driving HCC. In total, the screening library contained 290 gRNAs targeting 51 genes and 3 controls. After delivery of the plasmid library to the liver, the Myc gene provided a baseline level of oncogenic hyperproliferation, and the readout was gRNA prevalence pre- and post-proliferation via next-generation sequencing. We found that the combination of Myc and CRISPRa gRNA library led to a large number of expansive tumors in the liver as compared to Myc or the CRISPRa gRNA library only, which contained few to no tumors. gRNAs targeting control genes Ccnd1 and Vegfa were enriched as expected, validating our approach to annotate gene function during tumorigenesis. Additionally, we identified enrichment in gRNAs targeting genes Zbtb7b, Vps72, and Gba in multiple tumors, implicating a role in liver tumorigenesis. ZBTB7B and GBA expression levels are increased in patients with HCC recurrence as compared to patients in remission (data from BIOSTORM trial), while VPS72 is an unfavorable prognostic marker for HCC (data from The Human Protein Atlas). In this study, we have established a novel model of liver cancer that combines the Myc oncogene with a CRISPRa gRNA library to annotate driver genes in human HCC. Additionally, we identified several promising targets for the treatment of HCC. Ultimately, this study has the potential to impact on the care of patients with HCC in the US and worldwide. Citation Format: Alexandra Mariel Vázquez Salgado, Shirui He, Kirk J. Wangensteen. Identifying drivers of liver cancer using CRISPR activation screening in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 250.

One-step generation of mice with gene editing by Tol2 transposon-dependent gRNA delivery.

Conditional knockout mice are valuable tools for examining the functions of targeted genes in a time- and space-specific manner. Here, we generated gene-edited mice by using the Tol2 transposon to introduce guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-CRISPR-associated 9 (Cas9) mice, which express Cas9 in a Cre-dependent manner, with CAG-CreER mice. Transposase mRNA and plasmid DNA, which contained a gRNA sequence for the gene encoding tyrosinase flanked by the transposase recognition sequence, were injected together into fertilized eggs. As a result, the transcribed gRNA cleaved the target genome in a Cas9-dependent manner. Using this method, it is possible to generate conditional genome-edited mice more easily in a shorter period of time.

Plant breeding advancements with "CRISPR-Cas" genome editing technologies will assist future food security.

Genome editing techniques are being used to modify plant breeding, which might increase food production sustainably by 2050. A product made feasible by genome editing is becoming better known, because of looser regulation and widespread acceptance. The world's population and food supply would never have increased proportionally under current farming practices. The development of plants and food production has been greatly impacted by global warming and climate change. Therefore, minimizing these effects is crucial for agricultural production that is sustainable. Crops are becoming more resilient to abiotic stress because of sophisticated agricultural practices and a better understanding of the abiotic stress response mechanism. Both conventional and molecular breeding techniques have been used to create viable crop types both processes are time-consuming. Recently, plant breeders have shown an interest in genome editing approaches for genetic manipulation that use clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). To ensure the security of the food supply in the future, plant kinds with desired traits must be developed. A completely new era in plant breeding has begun because of the revolution in genome editing techniques based on the CRISPR/CRISPR-associated nuclease (Cas9) systems. All plants may effectively target a particular gene or group of loci using Cas9 and single-guide RNA (sgRNA). CRISPR/Cas9 can thereby save time and labor compared to conventional breeding methods. An easy, quick, and efficient method for directly altering the genetic sequences in cells is with the CRISPR and Cas9 systems. The CRISPR-Cas9 system, which was developed from components of the earliest known bacterial immune system, allows for targeted gene breakage and gene editing in a variety of cells/RNA sequences to guide endonuclease cleavage specificity in the CRISPR-Cas9 system. Editing can be directed to practically any genomic site by altering the guide RNA (gRNA) sequence and delivering it to a target cell along with the Cas9 endonuclease. We summarize recent CRISPR/Cas9 plant research findings, investigate potential applications in plant breeding, and make predictions about likely future breakthroughs and approaches to food security through 2050.

Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing.

A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon. The selection assay relies on hairpin substrates in which the target sequence is linked to partially randomized gRNAs in the same molecule, so that gRNA sequences that promote editing can be identified by sequencing. These RNA substrates were incubated in vitro with ADAR2 and the edited products were selected using amplification refractory mutation system PCR and used to regenerate the substrates for a new round of selection. After nine repetitions, hairpins which drove superior editing were identified. When gRNAs of these hairpins were delivered in trans, eight of the top ten short gRNAs drove superior editing both in vitro and in cellula. These results show that efficient small gRNAs can be selected using our approach, an important advancement for the clinical application of SDRE.

Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean

The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43-97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3' terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.

PAM-free cascaded strand displacement coupled with CRISPR-Cas12a for amplified electrochemical detection of SARS-CoV-2 RNA

The early diagnosis of coronavirus disease 2019 (COVID-19) is dependent on the specific and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Herein, we develop a highly sensitive and specific electrochemical biosensor for SARS-CoV-2 target RNA detection based on the integration of protospacer adjacent motif (PAM)-free cascaded toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a (PfTSDR-CRISPR). In this study, each target is transformed into multiple DNA substrates with bubble structure in the seed region by the cascaded TSDR, which can directly hybridize with guide RNA (gRNA) without PAM requirement and then activate CRISPR-Cas12a′s trans-cleavage activity. Subsequently, the hairpin DNA modified with methylene blue (MB-HP) is cleaved by activated CRISPR-Cas12a. Therefore, as MB leaves the electrode surface, a decreased current signal is obtained. With the involvement of PAM-free cascaded TSDRs and CRISPR-Cas12a amplification strategy, the PfTSDR-CRISPR-based electrochemical biosensor achieves the detection of target RNA as low as 40 aM. The biosensor has high sequence specificity, reliability and robustness. Thanks to the PAM-free cascaded TSDR, the biosensor can achieve universal detection of different target RNA without redesigning gRNA sequence of CRISPR-Cas12a. In addition, this biosensor successfully detects SARS-CoV-2 target RNA in complex samples, which highlights its potential for diagnosing COVID-19.

CRISPR genome editing using computational approaches: A survey.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has been widely used in various cell types and organisms. To make genome editing with Clustered regularly interspaced short palindromic repeats far more precise and practical, we must concentrate on the design of optimal gRNA and the selection of appropriate Cas enzymes. Numerous computational tools have been created in recent years to help researchers design the best gRNA for Clustered regularly interspaced short palindromic repeats researches. There are two approaches for designing an appropriate gRNA sequence (which targets our desired sites with high precision): experimental and predicting-based approaches. It is essential to reduce off-target sites when designing an optimal gRNA. Here we review both traditional and machine learning-based approaches for designing an appropriate gRNA sequence and predicting off-target sites. In this review, we summarize the key characteristics of all available tools (as far as possible) and compare them together. Machine learning-based tools and web servers are believed to become the most effective and reliable methods for predicting on-target and off-target activities of Clustered regularly interspaced short palindromic repeats in the future. However, these predictions are not so precise now and the performance of these algorithms -especially deep learning one's-depends on the amount of data used during training phase. So, as more features are discovered and incorporated into these models, predictions become more in line with experimental observations. We must concentrate on the creation of ideal gRNA and the choice of suitable Cas enzymes in order to make genome editing with Clustered regularly interspaced short palindromic repeats far more accurate and feasible.

In Vivo Crispr Screening Identifies Novel Vulnerabilities in ETO2/GLIS2-rearranged Leukemia

Background: CRISPR screening has allowed for massively parallel interrogation of the genome for vulnerabilities in a variety of cancer models. However, these experiments are largely performed in vitro due to the scale and conditions required to effectively conduct these screens. Furthermore, there still remain genetic subtypes of acute leukemia which are poorly represented among existing cell lines. We sought to determine the feasibility of conducting screens with focused libraries in patient-derived leukemia xenografts. Methods: We utilized a patient-derived acute megakaryoblastic leukemia (AMKL) bearing an ETO2/GLIS2 fusion, representing an aggressive subtype of pediatric acute leukemia with limited comparable cell lines (Gruber TA, et al. Cancer Cell 2012.) Based on its transplantability in NOD.scid.Il2Rγcnull-SGM3 (NSGS) mice vs culture in vitro, we performed all subsequent experiments in vivo. We first sought to establish stable Cas9 expression, which we successfully achieved by lentiviral transduction followed by FACS-enriched transplant, followed by a second round of FACS enrichment with limiting dilution transplant. As judged by mCherry flow cytometry, we achieved highly pure (>90%) Cas9-expressing populations, which we verified by western blotting. We further verified functionality of these cells by verifying cutting at the safe harbor locus AAVS1. In order to identify molecular vulnerabilities in this system, we identified the top 100 differentially expressed genes (DEG) between ETO2/GLIS2-rearranged leukemias versus other leukemia PDXs which we previously characterized. We included eight transcription factors with motifs enriched in our DEG analysis, as well as eight pan-essential positive controls (such as MYC, BRD4). Four guide RNAs (gRNA) per gene were selected from the Broad Brunello genome-wide library (Doench JG, et al. Nat Biotechnol 2016) as well as 50 "safe targeting" control guides (Morgens DW, et al. Nat Commun 2017) directed against non-essential genomic regions for a total of 518 guides. Guides were ordered as pooled oligonucleotides, cloned into a lentiviral gRNA backbone using standard techniques, then used to prepare lentivirus via transient transfection of 293T cells followed by titration to achieve an MOI of 0.3. With this MOI and an approximate engraftment frequency of 2% following intrafemoral injection, we transplanted 8.7e7 cells across eight NSGS mice to achieve adequate starting representation of our pooled library. Mice were sacrificed at humane endpoints, and spleens and bone marrow underwent PCR amplification for gRNA sequences followed by Illumina deep sequencing, and analysis with MAGeCK-VISPR (Wei L, et al. Genome Biol 2015). Results: One mouse per cohort was sacrificed at 24 hours post-transplant and sequenced for gRNA representation, which confirmed that library representation was largely preserved with our approach. At the time of sacrifice, spleen and bone marrow samples were independently sequenced, but no major bias in guide dropouts was observed between these compartments. Subsequent analyses were performed on the marrow alone. We observed the dropout of 38 genes with a false discovery rate < 0.25, including all positive control gRNAs as well as targets previously described to be essential through our own orthogonal experiments (BCL2) or previously published findings (GLIS2, GATA3) (Thirant C et al. Cancer Cell 2017). Among transcription factors found to be essential, we identified several which had not previously been strongly associated with acute leukemia (FOXD4L1, ZNF740). Conclusion: Patient-derived xenografts represent a unique platform for targeted functional genomic interrogation of acute leukemias. These screens can be performed with a tractable number of animals and modest sequencing requirements. Additional experiments are ongoing to identify the mechanistic implications of the vulnerabilities described here in ETO2/GLIS2-rearranged AMKL.

Polyvalent guide RNAs for CRISPR antivirals

CRISPR effector Cas13 recognizes and degrades RNA molecules that are complementary to its guide RNA (gRNA) and possesses potential as an antiviral biotechnology because it can degrade viral mRNA and RNA genomes. Because multiplexed targeting is a critical strategy to improve viral suppression, we developed a strategy to design of gRNAs where individual gRNAs have maximized activity at multiple viral targets, simultaneously, by exploiting the molecular biophysics of promiscuous target recognition by Cas13. These "polyvalent" gRNA sequences ("pgRNAs") provide superior antiviral elimination across tissue/organ scales in a higher organism (Nicotiana benthamiana) compared to conventionally-designed gRNAs-reducing detectable viral RNA by >30-fold, despite lacking perfect complementarity with either of their targets and, when multiplexed, reducing viral RNA by >99.5%. Pairs of pgRNA-targetable sequences are abundant in the genomes of RNA viruses, and this work highlights the need for specific approaches to the challenges of targeting viruses in eukaryotes using CRISPR.