Climate change and the diversity of consumer needs require innovative methods to continuously and rapidly modify existing crops for the development of new varieties.In the past decade genome editing ...by CRISPR/Cas and derivatives has emerged as a novel and effective technology for functional studies and gene discovery as well as for breeding new traits and genotypes.The development of novel CRISPR/Cas platforms, methods for the delivery of editing reagents, and methods for controlling gene regulation and detection of mutants have all expanded the scope of genome editing and other CRISPR/Cas-based approaches.
The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.
The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for ...engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR Cas9 technology offers.
CRISPR for neuroscientists Kalamakis, Georgios; Platt, Randall J.
Neuron (Cambridge, Mass.),
08/2023, Volume:
111, Issue:
15
Journal Article
Peer reviewed
Genome engineering technologies provide an entry point into understanding and controlling the function of genetic elements in health and disease. The discovery and development of the microbial ...defense system CRISPR-Cas yielded a treasure trove of genome engineering technologies and revolutionized the biomedical sciences. Comprising diverse RNA-guided enzymes and effector proteins that evolved or were engineered to manipulate nucleic acids and cellular processes, the CRISPR toolbox provides precise control over biology. Virtually all biological systems are amenable to genome engineering—from cancer cells to the brains of model organisms to human patients—galvanizing research and innovation and giving rise to fundamental insights into health and powerful strategies for detecting and correcting disease. In the field of neuroscience, these tools are being leveraged across a wide range of applications, including engineering traditional and non-traditional transgenic animal models, modeling disease, testing genomic therapies, unbiased screening, programming cell states, and recording cellular lineages and other biological processes. In this primer, we describe the development and applications of CRISPR technologies while highlighting outstanding limitations and opportunities.
In the primer by Kalamakis and Platt, they describe the development of CRISPR technologies and their applications in the diverse subfields of neuroscience. They also delve into outstanding limitations and emerging opportunities to investigate brain development, wiring, function, and history.
The CRISPR/Cas9 system development has revolutionized the field of genome engineering through the efficient creation of targeted breaks in the DNA of almost any organism and cell type, opening an ...avenue for a wide range of applications in biomedical research and medicine. Apart from gene edition through knock-in or knock-out approaches, CRISPR/Cas9 technology has been used for many other purposes, including regulation of endogenous gene expression, epigenome editing, live-cell imaging of chromosomal loci, edition of RNA and high-throughput screening. With all those technological improvements, CRISPR/Cas9 system has broadened the number of alternatives for studying gene function and the generation of more accurate disease models. Although many mechanistic questions remain to be answered and several challenges have yet to be addressed, the use of CRISPR/Cas9-based genome engineering technologies will increase our knowledge of disease processes and their treatment in the near future.
Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short ...Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.
Streptococci form a wide group of bacteria and are involved in both human and animal pathologies. Among pathogenic isolates, differences have been highlighted especially concerning their adaptation ...and virulence profiles. CRISPR-Cas systems have been identified in bacteria and many streptococci harbor one or more systems, particularly subtypes I-C, II-A, and III-A. Since the demonstration that CRISPR-Cas act as an adaptive immune system in
, a lactic bacteria, the diversity and role of CRISPR-Cas were extended to many germs and functions were enlarged. Among those, the genome editing tool based on the properties of Cas endonucleases is used worldwide, and the recent attribution of the Nobel Prize illustrates the importance of this tool in the scientific world. Another application is CRISPR loci analysis, which allows to easily characterize isolates in order to understand the interactions of bacteria with their environment and visualize species evolution. In this review, we focused on the distribution, diversity and roles of CRISPR-Cas systems in the main pathogenic streptococci.
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