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Zúñiga Orozco, Andrés. "Tecnología CRISPR-Cas9: una herramienta aplicable en la agricultura de Costa Rica". Repertorio Científico 20, n.º 2 (15 de noviembre de 2018): 131–38. http://dx.doi.org/10.22458/rc.v20i2.2396.
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En la actualidad la técnica de biología molecular CRISPRCas9, es la más estudiada a nivel global para la edición genómica. Por su facilidad, adaptabilidad, versatilidad, precisión y costo relativamente bajo es una tecnología que puede cambiar el panorama en la agricultura. El objetivo de este artículo es hacer una reseña y descripción acerca de la tecnología CRISPR-Cas9, mencionar algunas aplicaciones que se han realizado en la agricultura y citar algunas posibilidades que serían aplicables en Costa Rica.
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Barrangou, Rodolphe y David Bikard. "Guest editorial: CRISPRcas9: CRISPR-Cas systems: at the cutting edge of microbiology". Current Opinion in Microbiology 37 (junio de 2017): vii—viii. http://dx.doi.org/10.1016/j.mib.2017.09.015.
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Inam, Rafia. "Review Article on Anti-Microbial Resistance: Global Approach". Global Immunological & Infectious Diseases Review I, n.º I (30 de diciembre de 2016): 36–50. http://dx.doi.org/10.31703/giidr.2016(i-i).04.
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The rapid change in the failure of antimicrobial therapy due to an increase in the number of infections caused by antimicrobial resistance and the commencement of comprehensive resistant strains robustly claim detection of appliances causing the improvement of drug resistance. In this article, we review the double tactics to decrease antimicrobial resistance infections in hospitals by the improvement of hospital infection prevention and control (IPC) and Antimicrobial Stewardship (AMS) programs and their gradually significant part in decreasing the feast of antibiotic resistance using an ethical framework that enables clinicians and to appraise policies for rational use of an antibiotic in six practical steps. Though the procedure of bacteriophages and antibodies has been partially applied, further encouraging plans, such as probiotics and antimicrobial peptides, are under improvement. Novel approaches such as genetically modified phages and CRISPRCas9 also converse, and plasmid curing and anti-plasmid methods might reduce ARG existence and alert bacteria about antibiotics.
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Ophinni, Youdiil. "CRISPR-Cas9 as a curative drug for chronic viral infection". BIO Web of Conferences 41 (2021): 02010. http://dx.doi.org/10.1051/bioconf/20214102010.
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The innovation of CRISPR-Cas9 has single-handedly revolutionized biotechnology by enabling efficient and specific cutting of DNA. CRISPR-Cas9 approaches are promising not only to targetthe human genome but also DNA of pathogenic viruses, which coincidentally is the canonical function in its bacterial origin. Since 2014, a myriad of studies has proven the efficacy of CRISPR-Cas9 treatment to cleave viral DNA intermediates in vitro. One of the most widely targeted is theproviral genome of human immunodeficiency virus type-1 (HIV-1). The disease burden of HIV-1 is massive—the infection is incurable and has remained a pandemic for over four decades. Integrated HIV-1 provirus inside the human genome causes viral persistence inside latent cellular reservoirs, eluding antiretroviral therapy (ART) and sterilizing cure. Specific targeting anddisruption of HIV-1 proviral genome is necessary to achieve viral clearance, which can be achieved with CRISPR-Cas9. Here, we review the features and up to date evidence of CRISPRCas9 to target the HIV-1 proviral genome and suppress viral replication. We will also discuss potential CRISPR/Cas9 delivery methods in vivo, combination with other gene editing modalities and other therapeutic approaches, to bring gene editing-based HIV-1 cure closer into clinical use.
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Johnson, James, Franziska Linke, Cathy Merry y Beth Coyle. "MEDB-24. Tumour secreted extracellular matrix predicts survival and influences migration and cell death in SHH medulloblastoma 3D models". Neuro-Oncology 24, Supplement_1 (1 de junio de 2022): i110. http://dx.doi.org/10.1093/neuonc/noac079.398.
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Abstract Medulloblastoma is the most common malignant paediatric brain tumour. Four molecular sub-groups exist (WNT, Sonic hedgehog (SHH), Group 3 and Group 4), each associated with different patterns of metastasis and chemoresistance. We have shown that within these sub-groups further clinically relevant sub-types exist, characterised by differential expression of extracellular matrix (ECM) proteins. For example, good overall survival in two-thirds of SHH sub-group patients is associated with the expression of specific ECM proteins. Our aim here is to further characterise these ECM components in SHH medulloblastoma and to determine how they confer better overall survival in this sub-group. Using a combination of 3D OrbiSIMs and immunohistochemical staining we have identified, that when grown in 3D hyaluronic acid hydrogels or as spheroids, SHH medulloblastoma cell lines form an ECM shell-like structure composed of laminin, collagen and lumican. In addition, scRNAseq of SHH hydrogel nodules revealed sub-group specific clusters of cells with high levels of ECM interaction and adhesion. We therefore hypothesise that ECM interaction restricts SHH tumour invasion and metastasis through this shell-like structure. To understand how this ECM shell-like structure potentiates better survival in SHH medulloblastoma we have created a CRISPRcas9 laminin knockout SHH medulloblastoma cell line. 3D culture of the laminin knockout cell line demonstrated that laminin is essential for the formation of 3D cell structures as well as migration in SHH medulloblastoma. Furthermore, we have identified that apoptosis is increased in the laminin knockout SHH cell line, suggesting that apoptosis-targeted therapeutics may represent a beneficial treatment option for SHH patients whose tumours exhibit this ECM shell. In summary, tumour-secreted ECM plays a major role in SHH medulloblastoma progression. Expression of laminin, collagen or lumican can be used to classify SHH patients into low and high-risk groups with different therapeutic outcomes and treatment options.
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Dontenwill, M., M. Mercier, G. Gillmann, D. Reita, I. Lelong-Rebel, F. Noulet, A. Idbaih et al. "P11.59 Integrin a5 heterogeneous expression in glioblastoma is related to glioma stem cell subpopulations". Neuro-Oncology 21, Supplement_3 (agosto de 2019): iii57. http://dx.doi.org/10.1093/neuonc/noz126.205.
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Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive primary brain tumor. Treatment failure and recurrence are explained in part by tumoral heterogeneity. Our previous results showed that the integrin α5β1 is implicated in GBM aggressiveness and represents a relevant therapeutic target. Recently, we observed intra- and inter-tumor heterogeneity of integrin α5β1 expression. Heterogeneity may be linked to different glioma stem cell populations. MATERIAL AND METHODS Ten glioma stem cell lines were grown as neurospheres in stem cell medium and their differentiation was induced by serum and/or ATRA. Two cell lines (NCH421k and NCH644) were selected and were modified by depletion (CrisprCas9) or transfection of the α5 integrin gene. Polyclonal lines and individual clones were analyzed phenotypically in vitro, before and after differentiation, and in vivo in orthotopic xenografts of 2x104 cells in nude mice. TCGA datasets were used to validate the heterogeneous expression of α5 integrin in GBM. RESULTS TCGA data validate that α5 integrin mRNA was only over-expressed in the mesenchymal subclass of GBM. Our results show that α5 integrin protein is not expressed in stem cell culture conditions. However, α5 integrin expression is induced after differentiation in only half of the cell lines supporting the notion of tumoral heterogeneity of glioma stem cells. Interestingly, single cell-derived clone evaluation showed that intra-tumoral stem cell heterogeneity also exists at the level of α5 protein expression. When glioma stem cells are programmed or transduced to express α5 integrin, differentiated cells became more aggressive. Notably, they acquired a fibronectin-dependent motility and a proliferative phenotype. Interestingly, integrin α5 remained expressed in secondary stem cells obtained after dedifferentiation. The in vivo assays suggested that glioma stem cells, programmed to express the integrin, were prone to form larger tumors. CONCLUSION Our data support the hypothesis that some glioma stem cells are programmed to express the α5 integrin subunit in their differentiated progeny to form a more aggressive tumor. They add new evidences that both cell populations may be considered for new therapeutic strategies against GBM.
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Blatov, I. A., A. S. Shchurova, D. Yu Guschin, S. D. Zvereva y A. V. Popova. "CRISPR/Cas-Systems: characteristics and possibilities of use for editing bacterial genomes". Bacteriology 5, n.º 2 (2020): 38–48. http://dx.doi.org/10.20953/2500-1027-2020-2-38-48.
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CRISPR-Cas is the adaptive immune system of bacteria and archaea. Since 2012, when the first opportunity to use the CRISPR/Cas system for genome editing was realized, the number of studies in this area has been growing rapidly. Today, genomic editing to modify specific regions of the genomes of various organisms is considered one of the key methodologies of modern biology. This review is devoted to the history of discovery, classification, structure, operational mechanisms of CRISPRCas systems and strategies for editing the genomes of various bacterial species using this technology. Key words: genome editing, genome, CRISPR-Cas system, bacteria
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Lezon-Geyda, Kimberly, Vincent P. Schulz, Yelena Maksimova y Patrick G. Gallagher. "Altered Splicing from a Mutated Alternate Branch Point Is Common in Severe Alpha-Spectrin Linked Inherited Anemia". Blood 132, Supplement 1 (29 de noviembre de 2018): 503. http://dx.doi.org/10.1182/blood-2018-99-117752.
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Abstract Hereditary spherocytosis (HS), the most common inherited hemolytic anemia in Northern Europeans, is dominantly inherited in ~two third of cases. Clinically, patients with nondominant HS (ndHS) are more severely affected than those with typical, dominant HS. Biochemical and genetic studies implicate defects of α-spectrin in most ndHS patients and in patients with the related disorder hereditary pyropoikilocytosis (HPP). However, in most cases, neither the precise genetic basis nor the mechanism of disease are known. We studied individuals from 23 kindreds: 10 ndHS kindreds, 3 HPP kindreds, and 10 kindreds with transfusion-dependent (TD) anemia, using whole exome sequencing. A variety of novel mutant SPTA1 alleles were identified, including nonsense, splicing, and insertion/deletion mutations, frequently in trans to missense mutations. One patient had no SPTA1 mutations and 14 patients had only one SPTA1 mutation. Patients with 0 or 1 mutation all carried the common ndHS-linked α-spectrinBug Hill allele. We hypothesized that a production-defective SPTA1 allele is shared by these patients and is in linkage disequilibrium with the αBug Hill allele. Whole genome sequencing was performed on 2 ndHS patients heterozygous for the αBug Hill variant. Data were compared to samples in the 1000 Genomes database with the αBug Hill variant. A series of genetic analyses revealed a single common SPTA1 variant, the αLEPRA allele. This variant, described in a ndHS patient in trans to an SPTA1 nonsense mutation (JCI 98:2300, 1996), was associated with an elongated α-spectrin mRNA transcript. The αLEPRA allele was only present on 4 of 4610 alleles in 1000 Genomes. In all 4 cases, it was heterozygous and in cis to the αBugHill allele. Analysis of αBugHill haplotypes revealed 3 predominant patterns with the haplotype of the ndHS patients identical to the 4 heterozygous αLEPRA individuals. Genotyping the mutation-negative patient alleles revealed all carried the αLEPRA mutation. In the original report, RT-PCR of reticulocyte RNA demonstrated the αLEPRA allele was associated with an elongated α-spectrin mRNA transcript originating 70nt from the 3' end of intron 30. It was unclear if or how the αLEPRA mutation influenced α-spectrin mRNA splicing, as identical elongated α-spectrin mRNA transcripts are observed in erythroid cells from patients who do not carry the αLEPRA allele. Splicing analysis of intron 30 using SCROOGLE predicted: 1) a branch point at the expected location 31bp 5' of exon 31 (branch point 1, BP1); 2) an alternate upstream branch point centered on an "A" 2bp 3' of the αLEPRA mutation (BP2) 98bp 5' of exon 31; 3) identified a novel alternate 3' acceptor site downstream of BP2; and 4) the αLEPRA mutation significantly improves BP2. To determine if BP2 is a functional BP, we studied SPTA1 splicing using minigene assays in K562 cells. Wild type (WT) minigenes produced a small amount of elongated α-spectrin mRNA, while mutation of the obligate "A" of BP2 completely eliminated elongated transcript production. Mutation of the BP2 "A" using CRISPRCas9-based gene editing on a WT background in K562 cells eliminated the elongated α-spectrin mRNA transcripts. Minigene assays also revealed deletion of the alternate 3' acceptor splice site or improvement of BP1 to a U2 consensus site both eliminated the elongated transcript. Thus the αLEPRA mutation in intron 30, located upstream of an alternate 3' acceptor site, changes a weak alternate BP to a strong BP in the context of a poor primary BP. These changes lead to increased utilization of an alternate 3'acceptor site, creating an elongated transcript that leads to frameshift with a novel termination codon. This termination codon is in a position predicted to activate nonsense mediated decay (NMD). To address whether NMD of the elongated transcript is the mechanism of α-spectrin deficiency, we created K562 cells homozygous for the αLEPRA allele using gene editing and treated them with emetine or cycloheximide, NMD inhibitors. In both WT and homozygous αLEPRA cells, the total amount of elongated transcript increased relative to WT. These studies resolve an important unanswered question by demonstrating a novel mechanism of genetic disease is responsible for many cases of ndHS, HPP, and TD anemia. These data will facilitate disease diagnosis, as most current diagnostic gene panels do not include this intronic region, and they identify a new target for therapeutic gene manipulation. Disclosures No relevant conflicts of interest to declare.
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Iordache, Dumitrana, Gabriela-Maria Baci, Oana Căpriță, Anca Farkas, Andreea Lup y Anca Butiuc-Keul. "Correlation between CRISPR Loci Diversity in Three Enterobacterial Taxa". International Journal of Molecular Sciences 23, n.º 21 (23 de octubre de 2022): 12766. http://dx.doi.org/10.3390/ijms232112766.
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CRISPR-Cas is an adaptive immunity system of prokaryotes, composed of CRISPR arrays and the associated proteins. The successive addition of spacer sequences in the CRISPR array has made the system a valuable molecular marker, with multiple applications. Due to the high degree of polymorphism of the CRISPR loci, their comparison in bacteria from various sources may provide insights into the evolution and spread of the CRISPR-Cas systems. The aim of this study was to establish a correlation between the enterobacterial CRISPR loci, the sequence of direct repeats (DR), and the number of spacer units, along with the geographical origin and collection source. For this purpose, 3474 genomes containing CRISPR loci from the CRISPRCasdb of Salmonella enterica, Escherichia coli, and Klebsiella pneumoniae were analyzed, and the information regarding the isolates was recorded from the NCBI database. The most prevalent was the I-E CRISPR-Cas system in all three studied taxa. E. coli also presents the I-F type, but in a much lesser percentage. The systems found in K. pneumoniae can be classified into I-E and I-E*. The I-E and I-F systems have two CRISPR loci, while I-E* has only one locus upstream of the Cas cluster. PCR primers have been developed in this study for each CRISPR locus. Distinct clustering was not evident, but statistically significant relationships occurred between the different CRISPR loci and the number of spacer units. For each of the queried taxa, the number of spacers was significantly different (p < 0.01) by origin (Africa, Asia, Australia and Oceania, Europe, North America, and South America) but was not linked to the isolation source type (human, animal, plant, food, or laboratory strains).
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Wu, Mingming, Lixin Du, Ruizao Liu, Caihong Wei, Yayu Wang, Li Yang, Jiafan Liu, Yuqin Wang, Chuduan Wang y Xiaogang Wang. "Double-Muscled Phenotype in Mutant Sheep Directed by the CRISPRCas9 System". Cloning & Transgenesis 07, n.º 01 (2018). http://dx.doi.org/10.4172/2168-9849.1000161.
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Assefi,, Marjan, Sahar Asvadi, Hossein Ghahramani Almanghadim, Fatemeh Zeinali Sehrig, Zahra Foruzandeh, Shahab Masoumi y Hadis Sheikhi. "Potential Clinical Usefulness of CRISPR Cas9 Genome Editing in Cancer Treatment". Biomedical and Translational Science 1, n.º 2 (30 de junio de 2021). http://dx.doi.org/10.33425/2768-4911.1010.
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Cancer is still considered a main challenge regarding morbidity and mortality, although an earlier diagnosis through screening programs and more effective cure modalities have led to decreased mortality rates so the development of new genetic editing techniques such as zinc finger and TALENs or CRISPRCas9 has enabled the production of strong animal genetic models that sums up the cooperating oncogenic lesions influencing genes with a confirmed role in the proliferation and formation. This review presents the mechanisms of separate genome-editing approaches and explains each of the common nucleasebased platforms.
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O’Keeffe Ahern, Jonathan, Irene Lara-Sáez, Dezhong Zhou, Rodolfo Murillas, Jose Bonafont, Ángeles Mencía, Marta García et al. "Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system". Gene Therapy, 6 de agosto de 2021. http://dx.doi.org/10.1038/s41434-021-00282-6.
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AbstractRecent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.
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Mishra, Gauri, Lokesh Chandra Mishra, Kamakshi Srivastava, Juhi Rais, Manish Dixit y Vandana Kumari Singh. "CRISPR-Cas9: A Potent Gene-editing Tool for the Treatment of Cancer". Current Molecular Medicine 23 (13 de febrero de 2023). http://dx.doi.org/10.2174/1566524023666230213094308.
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Abstract: The prokaryotic adaptive immune system has clustered regularly interspaced short palindromic repeat. CRISPR-associated protein (CRISPR-Cas) genome editing systems have been harnessed. A robust programmed technique for efficient and accurate genome editing and gene targeting has been developed. Engineered cell therapy, in vivo gene therapy, animal modeling, and cancer diagnosis and treatment are all possible applications of this ground-breaking approach. Multiple genetic and epigenetic changes in cancer cells induce malignant cell growth and provide chemoresistance. The capacity to repair or ablate such mutations has enormous potential in the fight against cancer. The CRISPR-Cas9 genome editing method has recently become popular in cancer treatment research due to its excellent efficiency and accuracy. The preceding study has shown therapeutic potential in expanding our anticancer treatments by using CRISPR-Cas9 to directly target cancer cell genomic DNA in cellular and animal cancer models. In addition, CRISPR-Cas9 can combat oncogenic infections and test anticancer medicines. It may design immune cells and oncolytic viruses for cancer immunotherapeutic applications. In this review, these preclinical CRISPRCas9-based cancer therapeutic techniques are summarised, along with the hurdles and advancements in converting therapeutic CRISPR-Cas9 into clinical use. It will increase their applicability in cancer research.
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T Kondody, Rony, Manjusha Nambiar, Nandaprasad Shetty, Rajesh RNG y Nagaraj E. "Prospects of CRISPR-CAS 9 Gene Editing Technology in Dentistry: A Review". RGUHS Journal of Medical Sciences 13, n.º 1 (2023). http://dx.doi.org/10.26463/rjms.13_1_10.
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The recent advancement in molecular biology has revolutionized the field of medicine including various aspects of oral and craniofacial research. These revolutions in genetic engineering enabled the scientific community to understand details regarding gene functions and provide a platform for molecular cures. The latest gene-editing technology in this regard namely CRISPR-Cas system has opened a new era of versatile genetic manipulations because of its efficiency and ease of operation. Though this technology has enormous applications in different sections of the health care system this review focuses on explaining the prospect advances and insights regarding the future of the CRISPRCas system in dental practice.
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Pourcel, Christine, Marie Touchon, Nicolas Villeriot, Jean-Philippe Vernadet, David Couvin, Claire Toffano-Nioche y Gilles Vergnaud. "CRISPRCasdb a successor of CRISPRdb containing CRISPR arrays and cas genes from complete genome sequences, and tools to download and query lists of repeats and spacers". Nucleic Acids Research, 18 de octubre de 2019. http://dx.doi.org/10.1093/nar/gkz915.
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Abstract In Archaea and Bacteria, the arrays called CRISPRs for ‘clustered regularly interspaced short palindromic repeats’ and the CRISPR associated genes or cas provide adaptive immunity against viruses, plasmids and transposable elements. Short sequences called spacers, corresponding to fragments of invading DNA, are stored in-between repeated sequences. The CRISPR–Cas systems target sequences homologous to spacers leading to their degradation. To facilitate investigations of CRISPRs, we developed 12 years ago a website holding the CRISPRdb. We now propose CRISPRCasdb, a completely new version giving access to both CRISPRs and cas genes. We used CRISPRCasFinder, a program that identifies CRISPR arrays and cas genes and determine the system's type and subtype, to process public whole genome assemblies. Strains are displayed either in an alphabetic list or in taxonomic order. The database is part of the CRISPR-Cas++ website which also offers the possibility to analyse submitted sequences and to download programs. A BLAST search against lists of repeats and spacers extracted from the database is proposed. To date, 16 990 complete prokaryote genomes (16 650 bacteria from 2973 species and 340 archaea from 300 species) are included. CRISPR–Cas systems were found in 36% of Bacteria and 75% of Archaea strains. CRISPRCasdb is freely accessible at https://crisprcas.i2bc.paris-saclay.fr/.
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Voeneky, Silja. "New Challenges for International Standard Setting in Biomedicine: CrisprCas, Gene Drives, and How to Target Malaria". SSRN Electronic Journal, 2019. http://dx.doi.org/10.2139/ssrn.3391211.
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Boleij, Annemarie, Payam Fathi, William Dalton, Ben Park, Xinqun Wu, David Huso, Jawara Allen et al. "G-protein coupled receptor 35 (GPR35) regulates the colonic epithelial cell response to enterotoxigenic Bacteroides fragilis". Communications Biology 4, n.º 1 (14 de mayo de 2021). http://dx.doi.org/10.1038/s42003-021-02014-3.
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AbstractG protein-coupled receptor (GPR)35 is highly expressed in the gastro-intestinal tract, predominantly in colon epithelial cells (CEC), and has been associated with inflammatory bowel diseases (IBD), suggesting a role in gastrointestinal inflammation. The enterotoxigenic Bacteroides fragilis (ETBF) toxin (BFT) is an important virulence factor causing gut inflammation in humans and animal models. We identified that BFT signals through GPR35. Blocking GPR35 function in CECs using the GPR35 antagonist ML145, in conjunction with shRNA knock-down and CRISPRcas-mediated knock-out, resulted in reduced CEC-response to BFT as measured by E-cadherin cleavage, beta-arrestin recruitment and IL-8 secretion. Importantly, GPR35 is required for the rapid onset of ETBF-induced colitis in mouse models. GPR35-deficient mice showed reduced death and disease severity compared to wild-type C57Bl6 mice. Our data support a role for GPR35 in the CEC and mucosal response to BFT and underscore the importance of this molecule for sensing ETBF in the colon.
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Prakash, Aman y Manish Kumar. "Transcriptional analysis of CRISPR I-B arrays of Leptospira interrogans serovar Lai and its processing by Cas6". Frontiers in Microbiology 13 (29 de julio de 2022). http://dx.doi.org/10.3389/fmicb.2022.960559.
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In the genome of various Leptospira interrogans serovars, the subtype I-B locus of CRISPR-Cas possesses either one or multiple CRISPR arrays. In silico database (CRISPRCasdb) for predicting CRISPR-Cas reveals seven CRISPR arrays in L. interrogans serovar Lai positioned between the two independent cas-operons. Here, we present the redefined repeat-spacer boundaries of the CRISPR subtype I-B locus of serovar Lai. Such refinement of boundaries of arrays in serovar Lai was done after comparison with the characterized array of another serovar Copenhageni and the manual analysis of CRISPR flanking sequences. Using the reverse transcription-PCR (RT-PCR), we account that the seven CRISPR are transcriptionally active in serovar Lai. Our RT-PCR and quantitative real-time PCR analysis of transcripts in serovar Lai indicated that seven CRISPR of subtype I-B transcribe together as a single precursor unit. Moreover, the cleavage of the two miniature pre-crRNA of the subtype I-B by Cas6 demonstrates the biogenesis of the expected size of mature crRNA essential for the guided interference of foreign DNA. This study features insight into transcription direction and the crRNA biogenesis in serovar Lai essential for RNA-mediated interference of invading nucleic acids.