Cas9-Guide RNA Directed Genome Editing in Soybean.

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites.

CRISPR Genome Editing: Into the Second Decade

The Broad Institute Scores Another Victory in Its Battle with the University of California over the Patenting of CRIPSR

In It Together

Christmas disease, until now, has been considered incurable -a disease for life.

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

The emergence of an inexpensive and efficient tool for gene editing is accelerating the pace of cardiovascular research and beginning to show potential as a treatment tool. The new gene-editing technology leverages a bacterial defense mechanism against viral infections. Bacteria store sections of viral DNA in repeated sequences called CRISPR within their own genomes and use those sequences to identify and destroy viruses. Now, scientists are inserting genes they would like to target into CRISPR and pairing it with CAS9, an enzyme that cuts DNA. This process allows scientists to efficiently remove or replace a targeted gene. “It’s been a transformative technology,” said Kiran Musunuru, MD, PhD, MPH, an associate professor of cardiovascular medicine and genetics at the University of Pennsylvania’s Perelman School of Medicine. Gene editing has been a common tool in research for decades—particularly for producing model organisms with a particular genetic mutation. However, older techniques were more time-consuming, expensive, and more error-prone. CRISPR-CAS is allowing scientists to more easily and quickly produce model animals or human cell lines with specific genetic variants allowing experiments to proceed at an unprecedented pace or scale. Some preliminary studies have suggested that it also may be a useful tool for treating some forms of heart disease. The most immediate impact of CRISPR-CAS9 has been speeding the production …

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Rice (Oryza sativa L.) is one of the essential cereal crops for the majority of the world’s population. However, we need to ensure a continuous supply and enhanced the productivity of this crop in the purview of global climate change and increasing world population. Several crop improvement strategies, including genetic engineering and molecular breeding, have been routinely used to develop varieties superior in stress tolerance and yield. However, each one of them has limitations. Genome engineering or genome editing using targeted nucleases is recently being deployed as a key strategy to improve rice and other crops which promises a significant improvement in yield without the requirement of additional agricultural land in the future. Targeted genome editing using artificial nucleases has largely revolutionized the field of crops’ genome modification. Several studies recently used Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) to successfully modulate genes in a precise and predictable manner in plants for gene function studies and crop improvement programmes. These techniques open up new prospects to develop improved plant lines by adding important traits or by removing undesirable traits. The ability of these technologies to perform targeted and efficient modifications in genome sequence will undoubtedly lead to novel developments in plants, including crop plants. Moreover, due to the non-insertion of foreign DNA, this technique is socially acceptable and may help to alleviate regulatory issues associated with genetically modified plants. In this review, we describe the recent advancement in the CRISPR/Cas9 system and also highlight the strengths and weaknesses of this technology in comparison to the other two well-established genome editing platforms (ZFNs and TALENs). We have also discussed the small size new protein named CasX, its DNA cleavage characteristics, and its advantages over other CRISPR-Cas genome-editing enzymes. These technologies are mostly used for substitution of targeted gene fragments and insertion of exogenous DNA sequences into specific genomic location in crop plants that offer great potential for genetic improvement and breeding of rice.

The first gene therapy products for hemophilia A and B have recently been approved by the regulatory authorities. Although this is an important milestone for people with hemophilia, there is still a need to further improve on the efficacy, safety, and stability of expression and to ultimately include pediatric patients before the onset of arthropathy and other complications caused by uncontrolled bleeding. To overcome some of the limitations of conventional gene therapy strategies, gene editing is currently being explored in preclinical studies. Gene editing allows for targeted modifications of the human genome with unprecedented specificity based on zinc finger nuclease, meganuclease, transcription activator-like endonuclease, or clustered regularly interspaced short palindromic repeats (CRISPR) technologies that induce double-strand DNA breaks (DSB). Next-generation gene editing strategies, such as those dependent on CRISPR-derived base or prime editors, allow targeted genetic modification independent of the induction of DSBs, offering a potential safer alternative. Sustained efficacy and production of factor VIII or factor IX can be achieved after gene editing in patient-derived cells or in adult or newborn hemophilia A or B mouse models. These preclinical studies pave the way toward phase I/II clinical trials in patients with severe hemophilia. The potential risk of undesired off-target modifications of the human genome and adverse immune reactions, and the need for efficient delivery of the gene editing components, need to be rigorously addressed before the promise of gene editing for hemophilia can ultimately be fulfilled.

CRISPR/Cas9 system genome editing is revolutionizing genetics research in a wide spectrum of animal models in the genetic era. Among these animals, is the poultry species. CRISPR technology is the newest and most advanced gene-editing tool that allows researchers to modify and alter gene functions for transcriptional regulation, gene targeting, epigenetic modification, gene therapy, and drug delivery in the animal genome. The applicability of the CRISPR/Cas9 system in gene editing and modification of genomes in the avian species is still emerging. Up to date, substantial progress in using CRISPR/Cas9 technology has been made in only two poultry species (chicken and quail), with chicken taking the lead. There have been major recent advances in the modification of the avian genome through their germ cell lineages. In the poultry industry, breeders and producers can utilize CRISPR-mediated approaches to enhance the many required genetic variations towards the poultry population that are absent in a given poultry flock. Thus, CRISPR allows the benefit of accessing genetic characteristics that cannot otherwise be used for poultry production. Therefore CRISPR/Cas9 becomes a very powerful and robust tool for editing genes that allow for the introduction or regulation of genetic information in poultry genomes. However, the CRISPR/Cas9 technology has several limitations that need to be addressed to enhance its use in the poultry industry. This review evaluates and provides a summary of recent advances in applying CRISPR/Cas9 gene editing technology in poultry research and explores its potential use in advancing poultry breeding and production with a major focus on chicken and quail. This could aid future advancements in the use of CRISPR technology to improve poultry production.

Precise and efficient genome modifications present a great value in attempts to comprehend the roles of particular genes and other genetic elements in biological processes as well as in various pathologies. In recent years novel methods of genome modification known as genome editing, which utilize so called "programmable" nucleases, came into use. A true revolution in genome editing has been brought about by the introduction of the CRISP-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) system, in which one of such nucleases, i.e. Cas9, plays a major role. This system is based on the elements of the bacterial and archaeal mechanism responsible for acquired immunity against phage infections and transfer of foreign genetic material. Microorganisms incorporate fragments of foreign DNA into CRISPR loci present in their genomes, which enables fast recognition and elimination of future infections. There are several types of CRISPR-Cas systems among prokaryotes but only elements of CRISPR type II are employed in genome engineering. CRISPR-Cas type II utilizes small RNA molecules (crRNA and tracrRNA) to precisely direct the effector nuclease - Cas9 - to a specific site in the genome, i.e. to the sequence complementary to crRNA. Cas9 may be used to: (i) introduce stable changes into genomes e.g. in the process of generation of knock-out and knock-in animals and cell lines, (ii) activate or silence the expression of a gene of interest, and (iii) visualize specific sites in genomes of living cells. The CRISPR-Cas-based tools have been successfully employed for generation of animal and cell models of a number of diseases, e.g. specific types of cancer. In the future, the genome editing by programmable nucleases may find wide application in medicine e.g. in the therapies of certain diseases of genetic origin and in the therapy of HIV-infected patients.

Context: The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system is a groundbreaking gene-editing tool that shows great promise for modifying genomes. Derived from prokaryotic adaptive immune defense mechanisms, this technique has been used in research on human diseases, demonstrating remarkable therapeutic potential. Through CRISPR, specific genetic mutations in patients can be corrected during gene therapy, offering a solution for treating diseases that were previously untreatable using conventional methods. This review explores the recent progress and future prospects of the CRISPR system, focusing on its applications in medicine and stem cell engineering. Special emphasis is placed on medical applications, the latest target design or analysis tools for genome editing, advancements in stem cell engineering, and associated innovations and challenges. Evidence Acquisition: This study reviewed articles indexed in ISI, SID, PubMed, and PubMed Central from 2007 to 2024. Results: Cas9, a key protein in CRISPR gene editing, is an endonuclease capable of targeting and cutting specific DNA sequences, guided by short RNA sequences. The gene editing process involves homology-directed repair (HDR), non-homologous end joining (NHEJ), and base editing pathways. Base editing, which modifies the epigenome without inducing DNA breaks, is gaining increasing attention. However, CRISPR still faces technical challenges, and the development of more efficient "super" CRISPR technology will likely require time. This article reviews the effectiveness, limitations, and applications of the CRISPR system. Conclusions: CRISPR/Cas9 tools allow for the creation of precise models, leading to more effective treatment options for patients.

Being a rare inherited retinal dystrophy, retinitis pigmentosa (RP) in Laurence–Moon–Bardet–Biedl syndrome (LMBBS) is typically described in case series and reports. In the current study,[1] the authors describe a detailed big data analysis of patients with RP which helps document the degree and progeression of vision loss in affected patients. It also helps depict in detail, associated systemic and ocular co-morbidities. Genetic testing in such patients helps significantly. The advantages are threefold. First, we are able to share the likely course and progression of vision loss to patients by comparing them with people reported to have similar mutations. Second, the inheritance pattern can be informed and discussed with patients, which helps in family planning. Last but not the least, knowing the mutation helps us discuss potential ongoing gene therapy clinical trials and therapies in development, if available, for the same mutation. That said, in a busy retina practice, often explaining the advantages of the same and discussing the nuances around it may not always be possible. Further, the genomic sequencing technology is financially formidable for most patients, restricting its applicability at present. LMBBS is caused by mutations in the BBS gene (BBS1–BBS21), which account for 80% of cases with a clinical diagnosis of Bardet Biedl Syndrome (BBS).[2] The BBS1 gene, which is the most commonly affected, is responsible for cilia formation in photoreceptors. The impact of BBS gene mutations is seen in ciliated cells. Therefore, photoreceptor changes accompany hearing loss and renal, gonadal, and intellectual dysfunction. Advances in gene therapy have received US Food and Drug Administration (FDA) approval for retinal dystrophy caused by RPE65 gene mutations (voretigene neparvovec), and clinical trials are in progress for various other mutations causing RP. However, targeting the BBS gene mutations via gene therapy has been found to be complex due to the structural stoichiometry of the BBSome protein.[3] Gene editing using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) received the Nobel Prize in 2020. This promising new technique allows for editing the mutation. The CRISPR effector molecules consist of a guide RNA and Cas9 protein. The guide RNA identifies the target DNA to be edited, and the Cas9 protein cleaves the DNA, following which DNA repair mechanisms take place, which can insert the desired DNA bases, thereby correcting the mutation.[4] The technique can, however, cause DNA editing at other areas of the genome, called off-target effects. To mitigate this, CRISPR base editing and prime editing have been developed.[5] The technology holds promise and is currently in clinical trials for mutations in the CEP290 gene causing RP, the Brilliance Trial.[6] The role of CRISPR is yet to be explored in BBS gene mutations and perhaps could form a prudent area to explore in the future. To conclude, LMBBS is an important syndrome affecting multiple organs and tissues, the retina being one of them. Such descriptive studies driven by big data analytics help elucidate clinical characteristics of rare inherited retinal dystrophies. In future, an associated relevant genetic analysis of the causative mutations will help describe the mutational landscape in our country and plan therapies for the same.

Chapter 6 - Targeted genome editing: a new era in molecular biology