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Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice.
Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a small artificial intron to conditionally knockout (KO) a gene of interest in mice. However, many other labs are having difficulty getting the technique to work. The key problem appears to be either a failure in achieving correct splicing after the introduction of the artificial intron into the gene or, just as crucial, insufficient functional KO of the gene's protein after Cre-induced removal of the intron's branchpoint. Presented here is a guide on how to choose an appropriate exon and where to place the recombinase-regulated artificial intron (rAI) in that exon to prevent disrupting normal gene splicing while maximizing mRNA degradation after recombinase treatment. The reasoning behind each step in the guide is also discussed. Following these recommendations should increase the success rate of this easy, new, and alternative technique for producing tissue-specific KO mice.
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High-Throughput Generation of Pichia pastoris Knock-Out Strains by Using CRISPR/Cas9.
The CRISPR/Cas9 (CRISPR is an acronym for clustered regularly interspaced short palindromic repeats) system is a powerful molecular biological tool simplifying the process of genome engineering. Originally demonstrated to function in human and mouse cells, the portfolio of organisms that can be engineered by the new and groundbreaking technology was soon expanded. In the past years, CRISPR/Cas9 tools for use in Komagataella phaffii were reported to allow the generation of K. phaffii mutant strains in less than 2 weeks. In addition, the K. phaffii tailored system uses episomal vectors for the expression of the CRISPR/Cas9 elements, which allows the recycling of the plasmid after the CRISPR editing to obtain empty mutant strains. This means that the engineered strains do not carry the expression cassette of the resistance marker and CRISPR/Cas9 plasmid in their genome and are therefore a superb starting point for further investigations.In this chapter, we describe a pipeline for the high-throughput generation of K. phaffii mutant strains with interrupted open reading frames of genes, by using the CRISPR/Cas9 system in combination with error-prone repair of the double-strand break by NHEJ. The pipeline we developed consists of four steps: (a) CRISPR/Cas9 plasmids assembly, (b) transformation of K. phaffii, (c) screening for mutant strains, and (d) plasmid elimination and is due to the detailed description of every step being easily reproducible. To intensify and simplify the research work, most of the described procedures can be performed in a 96-well format.
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IndelQuant: A Workflow for Cost-Efficient Amplicon Sequencing Analysis of Genome-Edited Chickens.
Accurate quantification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-induced insertions and deletions (indels) is essential in chicken genome-editing studies, where low editing efficiencies and mosaicism can complicate the assessment of target-site modifications. Existing assays often involve a trade-off between affordability and the sensitivity orsequence information needed for detailed analysis. Here, we introduce IndelQuant, a cost-efficient workflow integrating sample-specific barcode PCR, pooled amplicon library construction, AdapterRemoval3-based read processing, and CRISPResso2-based indel quantification. The workflow provides graphical and tabularsummaries of editing efficiency, indel positions, indel-size distributions, and sample-level mutation profiles. We evaluated the workflow in TYR-edited DF-1 cells and genome-edited chickens. In edited chickens, IndelQuant detected tissue-specific indel signals, most below 5%, while negative controls showed background frequencies from 0 to 0.03%. These findings indicate that IndelQuant retains the sequence-level information provided by amplicon sequencing while simplifying sample processing and analysis, offering a practical approach to routine screening for low-frequency and mosaic indels in poultry genome-editing studies.
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Gene therapy strategies in tissue engineering: advances and applications for regenerative therapy.
Gene therapy has emerged as a promising strategy for tissue regeneration, offering the potential to address the limitations of conventional treatments. In this review we present an overview of applications of gene therapy in tissue regeneration, emphasizing recent advancements and future directions. Our work addresses gaps in the current literature by examining developments in molecular biology and genetics, such as clustered regularly interspaced short palindromic repeats (CRISPR) gene editing, advances in 3D bioprinting, and progress in gene delivery for tissue engineering. We describe case studies and clinical trials that demonstrate the potential of gene therapy applications in tissue engineering. We conclude by highlighting challenges and future directions, including emerging technologies and personalized gene-based approaches for tissue engineering research.
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Current trends in application of CRISPR/Cas9 in gene editing and diagnostics in Neglected tropical diseases (NTDs).
Neglected tropical diseases (NTDs) include more than a dozen of diseases which despite their fatality receive less attention from the research community worldwide. High cost diagnosis of these diseases and lack of trained community which can accurately interpret them is the major drawback in the healthcare system. Nowadays, in the genetic engineering era more emphasis is given to the modern gene editing tools such as Transcription Activator-Like Effector Nucleases (TALENS), Zinc Finger Nucleases (ZFNs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) due to their unique tailoring molecular machinery. This review article details the applicability of CRISPR/Cas9 as a modern gene editing tool in case of NTD parasites such as trypanosomatids with an aim to target their virulent genes. It has been observed through a number of studies that knocking in/out virulent genes of these parasites have led to a significant decrease in infectivity, growth rates along with morphological defects. The article also mentions various advanced CRISPR/Cas based diagnostics such as Specific High-Sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) and SHERLOCK4HAT which can detect parasite concentration as low as 2 attomolar/L (aM: 10- 18) and 1 parasite/µL respectively. This review also enlists various regulatory and biosafety issues, for example ecological imbalance which can arise as a consequence of CRISPR/Cas based gene drives employed to target parasitic vectors. Despite its wide applications, CRISPR/Cas is associated with several limitations like off-target effects and ecological imbalance to name a few.
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Novel Microbial Modification Tools to Convert Lipids into Other Value-Added Products.
CRISPR-Cas9 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) is a microbial adaptive immune system that has revolutionized the field of molecular biology and genome engineering. The Type II CRISPR system consists of Cas9 nuclease of Streptococcus pyogenes and the RNA complex that guides Cas9 nuclease to a specific sequence of DNA in the genome. The CRISPR-Cas9 technology has reformed our ability to edit DNA and to regulate expression levels of genes of interest to high precision and accuracy. It is a powerful technology, which is used for genome engineering of a wide range of organisms for various applications. Here, we describe a method involving CRISPR-Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms for biotechnological applications in yeast. The complete procedure of genome editing including target sequence selection, cloning gRNA with a target sequence, transformation, and verification of the desired mutation/deletion or insertion can be achieved within 2-3 weeks in yeast.
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Gene editing of hematopoietic stem cells: applications and advances.
Allogeneic hematopoietic stem cell transplantation remains the standard treatment for various hematologic genetic disorders resulting from single or multiple genes. However, this strategy is hindered by two main problems: failure to find a matching donor and the risk of graft-versus-host disease (GVHD) after transplantation. Recent advances in gene editing, particularly nucleases exemplified by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) and related derivative tools, have overcome the limitations posed by the poor specificity of traditional gene modification techniques. A robust groundwork has been established for developing efficient, precise, and diverse gene editing strategies, facilitating the clinical application of ex vivo modified autologous hematopoietic stem cells (HSCs). In contrast, autologous HSC transplantation does not have the previously mentioned problems associated with allogeneic transplantation. Consequently, gene editing involving ex vivo genetic modification of HSCs and subsequent reinfusion in a single patient has emerged, with related research progressing from investigation into fundamental mechanisms and proof-of-concept studies to clinical trials.
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Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum.
Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.
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A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum.
BACKGROUND: DNA-free, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing is a simple, convincing, and promising tool for precision crop breeding. The efficacy of designed CRISPR-based genome editing tools is a critical prerequisite for successful precision gene editing in crops. RESULTS: This study demonstrates that soil-grown leaf- or callus-derived pepper protoplasts are a useful system for screening of efficient guide RNAs for CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). CRISPR/Cas9 or Cpf1 were delivered as CRISPR/RNP complexes of purified endonucleases mixed with the designed single guide RNA, which can edit the target gene, CaMLO2 in two pepper cultivars with whole genome sequenced, Capsicum annuum 'CM334' and C. annuum 'Dempsey'. The designed guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) are conserved for CaMLO2 in both CM334 and Dempsey and cleave CaMLO2 in vitro. CRISPR/Cas9- or /Cpf1-RNP complexes were transfected into purely isolated protoplasts of the hot pepper CM334 and sweet pepper Dempsey by PEG-mediated delivery. Targeted deep sequencing analysis indicated that the targeted CaMLO2 gene was differentially edited in both cultivars, depending on the applied CRISPR/RNPs. CONCLUSIONS: Pepper protoplast-based CRISPR guide-RNA selection is a robust method to check the efficacy of designed CRISPR tools and is a prerequisite for regenerating edited plants, which is a critical time-limiting procedure. The rapid and convincing selection of guide RNA against a target genome reduces the laborious efforts for tissue culture and facilitates effective gene editing for pepper improvement.
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Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy.
Clustered regularly interspaced short palindromic repeats (CRISPR)-based HIV-1 genome editing has shown promising outcomes in in vitro and in vivo viral infection models. However, existing HIV-1 sequence variants have been shown to reduce CRISPR-mediated efficiency and induce viral escape. Two metrics, global patient coverage and global subtype coverage, were used to identify guide RNA (gRNA) sequences that account for this viral diversity from the perspectives of cross-patient and cross-subtype gRNA design, respectively. Computational evaluation using these parameters and over 3.6 million possible 20-bp sequences resulted in nine lead gRNAs, two of which were previously published. This analysis revealed the benefit and necessity of considering all sequence variants for gRNA design. Of the other seven identified novel gRNAs, two were of note as they targeted interesting functional regions. One was a gRNA predicted to induce structural disruption in the nucleocapsid binding site (Ψ), which holds the potential to stop HIV-1 replication during the viral genome packaging process. The other was a reverse transcriptase (RT)-targeting gRNA that was predicted to cleave the subdomain responsible for dNTP incorporation. CRISPR-mediated sequence edits were predicted to occur on critical residues where HIV-1 has been shown to develop resistance against antiretroviral therapy (ART), which may provide additional evolutionary pressure at the DNA level. Given these observations, consideration of broad-spectrum gRNAs and cross-subtype diversity for gRNA design is not only required for the development of generalizable CRISPR-based HIV-1 therapy, but also helps identify optimal target sites.
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