Cas9 Endonuclease Research Articles

CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells.

High-throughput CRISPR guide RNA (gRNA) library screen, that is, CRISPR/Cas9 screen, enables the unbiased identification of gene functions in a variety of biological processes. Typical pooled CRISPR/Cas9 screen couples a gRNA library and a guided Cas9 or dCas9 endonuclease to target specific gene loci, and then systematically uncover the causal link between candidate genes and observed cellular phenotypes via gRNA depletion or enrichment in screens. Here, we describe a detailed method of puromycin (PURO) concentration titration and lentiviral CRISPR gRNA library titration in Cas9 expressing monoclonal human iPSC line (Cas9+MNhiPSC) prior to performing the screens, conducting pooled CRISPR gRNA library screens in Cas9+MNhiPSC, genomic DNA extraction from the selected cell subpopulation and sequencing library preparation as well as next generation sequencing (NGS) to generate gRNA read counts. In CRISPR/Cas9 screen, we aim for 30% transduction efficiency (i.e., multiplicity of infection=0.3) to ensure most of infected cells receive only one gRNA. The principles in this method can be applied to CRISPR perturbation (knockout, activation, repression or base editing) screens with other CRISPR gRNA libraries across many other cell models and other species.

CRISPR-Mediated Genome Engineering in Aedes aegypti.

CRISPR-mediated genome engineering technologies have been adapted to a wide variety of organisms with high efficiency and specificity. The yellow fever mosquito, Aedes aegypti , is one such organism. It is also responsible for transmitting a wide variety of deadly viruses including Dengue, Zika, Yellow fever, and Chikungunya. The key to successful CRISPR-mediated gene editing applications is the delivery of both Cas9 ribonuclease and single-guide RNA (sgRNA ) to the nucleus of desired cells. Various methods have been developed for supplying the Cas9 endonuclease, sgRNA , and donor DNA to Ae. aegypti. In this chapter, we focus on methods of direct embryo delivery of editing components, presenting detailed step-by-step CRISPR/Cas9-based genome-editing protocols for inducing desired heritable edits in mosquitoes as well as insights into successful application of these protocols. We also highlight potential opportunities for customizing these protocols to manipulate the mosquito genome for innovative in vivo gene function studies.

Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions.

Investigating the process of gamete formation in plants often requires the use of mutants of selected genes in various genetic backgrounds. For example, analysis of meiotic recombination based on sequencing or genotyping requires the generation of hybrids between two lines. Although T-DNA mutant collections of Arabidopsis thaliana are vast and easily accessible, they are largely confined toCol-0 background. This chapter describes how to efficiently generate knock-out mutants in different Arabidopsis accessions using CRISPR/Cas9 technology. The presented system is based on designing two single-guide RNAs (sgRNAs), which direct the Cas9 endonuclease to generate double-strand breaks at two sites, leading to genomic deletion in targeted gene. The presence of seed-expressed dsRed fluorescence cassette in the CRISPR construct facilitates preselection of genome-edited and transgene-free plants by monitoring the seed fluorescence under the epifluorescent microscope. The protocol provides the detailed information about all steps required to perform genome editing and to obtain loss-of-function mutants in different Arabidopsis accessions within merely two generations.

Optimized Proteolistic Protocol for the Delivery of the Cas9 Protein in Phaeodactylum tricornutum.

The CRISPR/Cas9 system coupled with proteolistics is a DNA-free nuclear transformation method based on the introduction of ribonucleoprotein (RNP) complexes into cells. The method has been set up for diatoms as an alternative to genetic transformation via biolistics and has the advantages of reducing off-target mutations, limiting the working time of the Cas9 endonuclease, and overcoming the occurrence of random insertions of the transgene in the genome. We present a point-by-point description of the protocol with modifications that make it more cost-effective, by reducing the amount of the enzyme while maintaining a comparable efficiency to the original protocol, and with an increased concentration of the selective drug which allows to reduce false positives.

Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9.

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.

Alteration of Salmonella enterica Virulence and Host Pathogenesis through Targeting sdiA by Using the CRISPR-Cas9 System.

Salmonella enterica is a common cause of many enteric infections worldwide and is successfully engineered to deliver heterologous antigens to be used as vaccines. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 endonuclease is a promising genome editing tool. In the current study, a CRISPR-Cas9 system was used to target S. enterica sdiA that encodes signal molecule receptor SdiA and responds to the quorum sensing (QS) signaling compounds N-acylhomoserine lactones (AHLs). For this purpose, sdiA was targeted in both S. enterica wild type (WT) and the ΔssaV mutant strain, where SsaV has been reported to be an essential component of SPI2-T3SS. The impact of sdiA mutation on S. enterica virulence was evaluated at both early invasion and later intracellular replication in both the presence and absence of AHL. Additionally, the influence of sdiA mutation on the pathogenesis S. enterica WT and mutants was investigated in vivo, using mice infection model. Finally, the minimum inhibitory concentrations (MICs) of various antibiotics against S. enterica strains were determined. Present findings show that mutation in sdiA significantly affects S. enterica biofilm formation, cell adhesion and invasion. However, sdiA mutation did not affect bacterial intracellular survival. Moreover, in vivo bacterial pathogenesis was markedly lowered in S. enterica ΔsdiA in comparison with the wild-type strain. Significantly, double-mutant sdiA and ssaV attenuated the S. enterica virulence and in vivo pathogenesis. Moreover, mutations in selected genes increased Salmonella susceptibility to tested antibiotics, as revealed by determining the MICs and MBICs of these antibiotics. Altogether, current results clearly highlight the importance of the CRISPR-Cas9 system as a bacterial genome editing tool and the valuable role of SdiA in S. enterica virulence. The present findings extend the understanding of virulence regulation and host pathogenesis of Salmonella enterica.

Targeted insertion of large genetic payloads using cas directed LINE-1 reverse transcriptase

A difficult genome editing goal is the site-specific insertion of large genetic constructs. Here we describe the GENEWRITE system, where site-specific targetable activity of Cas endonucleases is coupled with the reverse transcriptase activity of the ORF2p protein of the human retrotransposon LINE-1. This is accomplished by providing two RNAs: a guide RNA targeting Cas endonuclease activity and an appropriately designed payload RNA encoding the desired insertion. Using E. coli as a simple platform for development and deployment, we show that with proper payload design and co-expression of helper proteins, GENEWRITE can enable insertion of large genetic payloads to precise locations, although with off-target effects, using the described approach. Based upon these results, we describe a potential strategy for implementation of GENEWRITE in more complex systems.

The Alzheimer's disease risk gene CD2AP modulates mammalian synaptic structure and plasticity

CD2-Associated protein (CD2AP) is associated with increased risk of late-onset Alzheimer's disease. We have previously shown that loss of CD2AP's Drosophila homolog, cindr, exacerbates Tau toxicity and impairs synaptic maturation and function at the neuromuscular junction. However, little is understood about CD2AP at the central mammalian synapse. The present study demonstrates conservation of CD2AP function at the pre-synapse from fly to mouse, as well as characterizes CD2AP's role in maintaining post-synaptic structure and function.We examine short- and long-term plasticity using electrophysiologic measurements and synaptic structure and morphology using confocal imaging. Experiments were performed on homozygous CD2AP knockout mice and heterozygous CD2AP knockout mice to investigate potential haploinsufficient requirement. Mice constitutively expressing Cas9 endonuclease were used for brain-specific CD2AP knockout.Homozygous and heterozygous CD2AP knockout mice exhibit increased paired-pulse facilitation, a form of short-term plasticity. This matches data from Drosophila, implying conservation from fly to mouse, and indicates decreased probability of presynaptic vesicle release with CD2AP loss. We found no change in long-term potentiation (LTP) at 4 to 6 weeks; however, our preliminary experiments demonstrate decreased LTP in aged heterozygous CD2AP knockouts. Ongoing experiments are investigating age as a factor in CD2AP function. Lastly, CD2AP knockout neurons exhibit increased dendritic branching and homozygous knockout mice have increased expression of postsynaptic density protein 95.Our analyses implicate CD2AP in modulation of synaptic plasticity and structure, suggest a conservation of CD2AP function from flies to mice, and support CD2AP haploinsufficiency. Going forward, we will investigate how CD2AP loss affects existing Alzheimer's pathogenesis in a P301S tauopathy mouse model.

Targeted Knockout of Eukaryotic Translation Initiation Factor 4E Confers Bymovirus Resistance in Winter Barley.

The Eukaryotic Translation Initiation Factor 4E (EIF4E) is a well-known susceptibility factor for potyvirus infections in many plant species. The barley yellow mosaic virus disease, caused by the bymoviruses Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), can lead to yield losses of up to 50% in winter barley. In autumn, the roots of young barley plants are infected by the soil-borne plasmodiophoraceous parasite Polymyxa graminis L. that serves as viral vector. Upon viral establishment and systemic spreading into the upper parts of the plants, yellow mosaics occur as first symptoms on leaves. In the further course of plant development, the disease entails leaf necrosis and increased susceptibility to frost damage. Thanks to the rym4 and rym5 allelic variants of the HvEIF4E gene, more than two thirds of current European winter barley cultivars are resistant to BaYMV and BaMMV. However, several strains of BaYMV and BaMMV have already overcome rym4- and rym5-mediated resistance. Accordingly, new resistance-conferring alleles are needed for barley breeding. Therefore, we performed targeted mutagenesis of the EIF4E gene by Cas9 endonuclease in BaMMV/BaYMV-susceptible winter barley cv. “Igri”. Small insertions were generated, resulting in a shift of the translational reading frame, thereby causing the loss-of-function of EIF4E. The mutations occurred in the homozygous state already in the primary mutants. Their progeny proved invariably homozygous and fully resistant to mechanical inoculation with BaMMV. EIF4E knockout plants showed normal growth habit and produced grains, yet exhibited a yield penalty.

Cell erythroid maturation approach: a new paradigm in the road map towards a cure for β-thalassaemia syndromes.

Cell erythroid maturation approach: a new paradigm in the road map towards a cure for β-thalassaemia syndromes.

Establishment of a pig CRISPR/Cas9 knockout library for functional gene screening in pig cells.

As an important farm animal, pig functional genomic study can help understand the molecular mechanism related to the key economic traits of pig, such as growth, reproduction, or disease. The genome-scale library based on clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system facilitates discovery of key genes involved in a specific function or phenotype, allowing for an effective "phenotype-to-genotype" strategy for functional genomic study. We designed and constructed a pig genome-scale CRISPR/Cas9 knockout library targeting 16,888 genes with 970,001 unique sgRNAs. The library is a single-vector system including both Cas9 and sgRNA, and packaged into lentivirus for an easy cell delivery for screening. To establish a screening method in pig cells, we used diphtheria toxin (DT)-induced cell death as a model to screen the host genes critical for DT toxicity in pig PK-15 cells. After lentiviral transduction and two sequential screening with DT treatment, the highest-ranking candidates we identified were previously validated genes, HBEGF, DPH1, DPH2, DPH3, DPH5, DNAJC24, and ZBTB17, which are DT receptor and the key factors involved in biosynthesis of diphthamide, the target of DT action. The function and gene essentiality of candidates were further confirmed by gene knockout and DT toxicity assay in PK-15 cells. Our CRISPR knockout library targeting pig whole genome establishes a promising platform for pig functional genomic analysis.

CRISPER/Cas in Plant Natural Product Research: Therapeutics as Anticancer and other Drug Candidates and Recent Patents.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated9 (Cas9) endonuclease system is a facile, highly efficient and selective site-directed mutagenesis tool for RNA-guided genome-editing. CRISPR/Cas9 genome-editing strategy uses designed guide-RNAs that recognizes a 3 base-pair protospacer adjacent motif (PAM) sequence in the target-DNA. CRISPR/Cas-editing tools have mainly been employed in crop plants in relation to yield and stress tolerance. However, the immense potential of this technology has not yet been fully utilized in medicinal plants in deciphering or modulating secondary metabolic pathways producing therapeutically active phytochemicals against cancer and other diseases. The present review elucidates the use of CRISPR-Cas9 as a promising genome-editing tool in plants and plant-derived natural products with anticancer and other therapeutic applications. It also includes recent patents on the therapeutic applications of CRISPR-CAS systems implicated to cancer and other human medical conditions. Popular search engines, such as PubMed, Scopus, Google Scholar, Google Patents, Medline, ScienceDirect, SpringerLink, EMBASE, Mendeley, etc., were searched in order to retrieve literature using relevant keywords viz. CRISPER/Cas, plant natural product research, anticancer, therapeutics, etc., either singly or in various combinations. Retrieved citations and further cross-referencing among the literature have resulted in a total number of 71 publications and 3 patents are being cited in this work. Information presented in this review aims to support further biotechnological and clinical strategies to be carried using CRISPER/ Cas mediated optimization of plant natural products against cancer and an array of other human medical conditions. Off late, knock-in and knock-out, point mutation, controlled tuning of gene-expression and targeted mutagenesis have enabled the versatile CRISPR/Cas-editing device to engineer medicinal plants' genomes. In addition, by combining CRISPR/Cas-editing tool with next-generation sequencing (NGS) and various tools of system biology, many medicinal plants have been engineered genetically to optimize the production of valuable bioactive compounds of industrial significance.

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin.

Translating the CRISPR/Cas9 genome editing technology into clinics is still hampered by rather unspecific, unsafe and/or inconvenient approaches for the delivery of its main components - the Cas9 endonuclease and a guide RNA - into cells. Here, we describe the development of a novel transient and non-viral Cas9 delivery strategy based on the translocation machinery of the Bacillus anthracis anthrax toxin, PA (protective antigen). We show that Cas9 variants fused to the N-terminus of the lethal factor or to a hexahistidine tag are shuttled through channels formed by PA into the cytosol of human cells. As proof-of-principle, we applied our new approach, denoted as CRISPA, to knock out lipolysis-stimulated lipoprotein receptor (LSR) in the human colon cancer cell line HCT116 and green-fluorescent protein (GFP) in human embryonic kidney 293T cells stably expressing GFP. Notably, we confirmed that the transporter PA can be adapted to recognize specific host cell-surface receptor proteins and may be optimized for cell type-selective delivery of Cas9. Altogether, CRISPA provides a novel, transient and non-viral way to deliver Cas9 into specific cells. Thus, this system is an additional step towards safe translation of the CRISPR/Cas9 technology into clinics.

Caracterización del complejo genético tracrARN-DRARN asociado al sistema CRISPR-Cas9 del fitosimbionte Acholeplasma palmae descriptor: interés biotecnológico

The CRISPR-Cas9 technology used in plant biotechnology is based on the use of Cas9 endonucleases to generate precise cuts in the genome, and a duplex consisting of a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (DRRNA) which are precursors of guide RNA (sgRNA) commercially redesigned (sgRNA-Cas9) to guide gene cleavage. Most of these tools come from clinical bacteria. However, there are several CRISPR-Cas9 systems in environmental microorganisms such as phytoendosymbionts of plants of the genus Acholeplasma. But the exploitation of these systems more compatible with plants requires using bioinformatics tools for prediction and study. We identified and characterized the elements associated with the duplex in the genome of A. palmae. For this, the protein information was obtained from the Protein Data Bank and the genomics from GenBank/NCBI. The CRISPR system was studied with the CRISPRfinder software. Alignment algorithms and NUPACK software were used to identify the tracrRNA and DRRNA modules, together with various computational software for genetic, structural and biophysical characterization. A CRISPR-Cas system was found in A. palmae with type II-C characteristics, as well as a thermodynamically very stable duplex, with flexible regions, exhibiting a docking power with Cas9 thermodynamically favored. These results are desirable in programmable gene editing systems and show the possibility of exploring native molecular tools in environmental microorganisms applicable to the genetic manipulation of plants, as more research is carried out. This study represents the first report on the thermodynamic stability and molecular docking of elements associated with the tracrRNA-DRRNA duplex in the phytosymbiont A. palmae.

Compendium of Plant-Specific CRISPR Vectors and Their Technical Advantages.

CRISPR/Cas mediated genome editing is a revolutionary approach for manipulating the plant genome. However, the success of this technology is highly dependent on selection of a specific vector and the other components. A plant-specific CRISPR/Cas vector usually consists of a Cas gene, target-specific gRNA, leader sequence, selectable marker gene, precise promoters, and other accessories. It has always been challenging to select the specific vector for each study due to a lack of comprehensive information on CRISPR vectors in one place. Herein, we have discussed every technical aspect of various important elements that will be highly useful in vector selection and efficient editing of the desired plant genome. Various factors such as the promoter regulating the expression of Cas and gRNA, gRNA size, Cas variants, multicistronic gRNA, and vector backbone, etc. influence transformation and editing frequency. For example, the use of polycistronic tRNA-gRNA, and Csy4-gRNA has been documented to enhance the editing efficiency. Similarly, the selection of an efficient selectable marker is also a very important factor. Information on the availability of numerous variants of Cas endonucleases, such as Cas9, Cas12a, Cas12b, Casɸ, and CasMINI, etc., with diverse recognition specificities further broadens the scope of editing. The development of chimeric proteins such as Cas fused to cytosine or adenosine deaminase domain and modified reverse transcriptase using protein engineering enabled base and prime editing, respectively. In addition, the newly discovered Casɸ and CasMINI would increase the scope of genetic engineering in plants by being smaller Cas variants. All advancements would contribute to the development of various tools required for gene editing, targeted gene insertion, transcriptional activation/suppression, multiplexing, prime editing, base editing, and gene tagging. This review will serve as an encyclopedia for plant-specific CRISPR vectors and will be useful for researchers.

Rapid Characterization of Complex Killer Cell Immunoglobulin-Like Receptor (KIR) Regions Using Cas9 Enrichment and Nanopore Sequencing.

Long-read sequencing approaches have considerably improved the quality and contiguity of genome assemblies. Such platforms bear the potential to resolve even extremely complex regions, such as multigenic immune families and repetitive stretches of DNA. Deep sequencing coverage, however, is required to overcome low nucleotide accuracy, especially in regions with high homopolymer density, copy number variation, and sequence similarity, such as the MHC and KIR gene clusters of the immune system. Therefore, we have adapted a targeted enrichment protocol in combination with long-read sequencing to efficiently annotate complex KIR gene regions. Using Cas9 endonuclease activity, segments of the KIR gene cluster were enriched and sequenced on an Oxford Nanopore Technologies platform. This provided sufficient coverage to accurately resolve and phase highly complex KIR haplotypes. Our strategy eliminates PCR-induced amplification errors, facilitates rapid characterization of large and complex multigenic regions, including its epigenetic footprint, and is applicable in multiple species, even in the absence of a reference genome.

Target Gene Identification and sgRNA Design for Waterlogging Tolerance in Foxtail Millet via CRISPR-Based Transcriptional Activation

Background: CRISPR activation (CRISPRa) uses non-functional Cas9 endonuclease (dCas9) but retains the genome targeting ability through its single guide RNAs (sgRNAs). CRISPRa is widely used as a gene activation system exploiting its ability in recruiting various transcriptional activation domains (TADs) for enhancing expression of the target gene(s). Drought tolerant and resource-efficient crops like millets have the potential for mitigating effects of climate change and for enhancing food security. Objective: This study aimed to use the Setaria italica (foxtail millet) genome sequence in the identification of a target gene and the subsequent generation of sgRNAs for use in CRISPRa for conferring water logging tolerance that will benefit the future expansion of its cultivation area. Methods and Results: Leveraging existing RNA-seq data and information on functional studies in model plants and from other cereal species, maize and barley, have enabled the identification of candidate ERFVII from the foxtail millet genome sequence in the attempt to engineer water logging tolerance. The study provides a step-by-step example for using publicly accessible databases and bioinformatics tools from NCBI and Phytozome for the identification and characterization of the ortholog from Setaria italica. Soft berry was used for promoter annotation to obtain the transcription start site (TSS). Subsequently, CRISP-P 2.0 design tools were employed to generate and select a few efficient sgRNAs for CRISPRa that minimize potentially deleterious off-target binding. Conclusion: The study is a useful example of how to advance in genomics research including the revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species, through the utilization of the available genome sequence.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

The comparison of ZFNs, TALENs, and SpCas9 by GUIDE-seq in HPV-targeted gene therapy

Zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR-associated Cas9 endonucleases are three major generations of genome editing tools. However, no parallel comparison about the efficiencies and off-target activity of the three nucleases has been reported, which is critical for the final clinical decision. We for the first time developed the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method in ZFNs and TALENs with novel bioinformatics algorithms to evaluate the off-targets. By targeting human papillomavirus 16 (HPV16), we compared the performance of ZFNs, TALENs, and SpCas9 in vivo. Our data showed that ZFNs with similar targets could generate distinct massive off-targets (287–1,856), and the specificity could be reversely correlated with the counts of middle “G” in zinc finger proteins (ZFPs). We also compared the TALENs with different N-terminal domains (wild-type [WT]/αN/βN) and G recognition modules (NN/NH) and found the design (αN or NN) to improve the efficiency of TALEN inevitably increased off-targets. Finally, our results showed that SpCas9 was more efficient and specific than ZFNs and TALENs. Specifically, SpCas9 had fewer off-target counts in URR (SpCas9, n = 0; TALEN, n = 1; ZFN, n = 287), E6 (SpCas9, n = 0; TALEN, n = 7), and E7 (SpCas9, n = 4; TALEN, n = 36). Taken together, we suggest that for HPV gene therapies, SpCas9 is a more efficient and safer genome editing tool. Our off-target data could be used to improve the design of ZFNs and TALENs, and the universal in vivo off-target detection pipeline for three generations of artificial nucleases provided useful tools for genome engineering-based gene therapy.

Wobble Editing of Cre-box by Unspecific CRISPR/Cas9 Causes CCR Release and Phenotypic Changes in Bacillus pumilus.

CRISPR-associated Cas9 endonuclease (CRISPR/Cas9) systems are widely used to introduce precise mutations, such as knocking in/out at targeted genomic sites. Herein, we successfully disrupted the transcription of multiple genes in Bacillus pumilus LG3145 using a series of unspecific guide RNAs (gRNAs) and UgRNA:Cas9 system-assisted cre-box editing. The bases used as gRNAs shared 30–70% similarity with a consensus sequence, a cis-acting element (cre-box) mediating carbon catabolite repression (CCR) of many genes in Bacillus. This triggers trans-crRNA:Cas9 complex wobble cleavage up/downstream of cre sites in the promoters of multiple genes (up to 7), as confirmed by Sanger sequencing and next-generation sequencing (NGS). LG3145 displayed an obvious CCR release phenotype, including numerous secondary metabolites released into the culture broth, ∼ 1.67 g/L white flocculent protein, pigment overflow causing orange-coloured broth (absorbance = 309 nm), polysaccharide capsules appearing outside cells, improved sugar tolerance, and a two-fold increase in cell density. We assessed the relationship between carbon catabolite pathways and phenotype changes caused by unspecific UgRNA-directed cre site wobble editing. We propose a novel strategy for editing consensus targets at operator sequences that mediates transcriptional regulation in bacteria.