CRISPR Nickase-Mediated Base Editing in Yeast.

The CRISPR/Cas9 system has been applied to efficient genome editing in many eukaryotic cells. However, the bases that can be edited by this system have been limited to those within the protospacer adjacent motif (PAM) and guide RNA-targeting sequences. In this study, we developed a genome-wide base editing technology, “CRISPR Nickase system” that utilizes a single Cas9 nickase. This system was free from the limitation of editable bases that was observed in the CRISPR/Cas9 system, and was able to precisely edit bases up to 53 bp from the nicking site. In addition, this system showed no off-target editing, in contrast to the CRISPR/Cas9 system. Coupling the CRISPR Nickase system with yeast gap repair cloning enabled the construction of yeast mutants within only five days. The CRISPR Nickase system provides a versatile and powerful technology for rapid, site-specific, and precise base editing in yeast.

Prime editors (PEs) enable targeted precise editing, including the generation of substitutions, insertions and deletions, in eukaryotic genomes. However, their genome-wide specificity has not been explored. Here, we developed Nickase-based Digenome-seq (nDigenome-seq), an in vitro assay that uses whole-genome sequencing to identify single-strand breaks induced by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nickase. We used nDigenome-seq to screen for potential genome-wide off-target sites of Cas9 H840A nickase, a PE component, targeted to nine human genomic sites. Then, using targeted amplicon sequencing of off-target candidates identified by nDigenome-seq, we showed that only five off-target sites showed detectable PE-induced modifications in cells, at frequencies ranging from 0.1 to 1.9%, suggesting that PEs provide a highly specific method of precise genome editing. We also found that PE specificity in human cells could be further improved by incorporating mutations from engineered Cas9 variants, particularly eSpCas9 and Sniper Cas9, into PE.

Exogenous DNA can be a template to precisely edit a cell’s genome. However, the delivery of in vitro-produced DNA to target cells can be inefficient, and low abundance of template DNA may underlie the low rate of precise editing. One potential tool to produce template DNA inside cells is a retron, a bacterial retroelement involved in phage defense. However, little effort has been directed at optimizing retrons to produce designed sequences. Here, we identify modifications to the retron non-coding RNA that result in more abundant reverse transcribed DNA. By testing architectures of the retron operon that enable efficient reverse transcription, we find that gains in DNA production are portable from prokaryotic to eukaryotic cells and result in more efficient genome editing. Finally, we show that retron RT-DNA can be used to precisely edit cultured human cells. These experiments provide a general framework to produce DNA using retrons for genome modification.

RNA-Guided Genome Editing in Plants Using a CRISPR–Cas System

The potential to therapeutically alter the genome is one of the remarkable scientific developments in recent years. Genome editing technologies have provided an opportunity to precisely alter genomic sequence(s) in eukaryotic cells as a treatment option for various genetic disorders. These technologies allow the correction of harmful mutations in patients by precise nucleotide editing. Genome editing technologies such as CRISPR (clustered regularly interspaced short palindromic repeat) and base editors have greatly contributed to the practical applications of gene editing. However, these technologies have certain limitations, including imperfect editing, undesirable mutations, off-target effects, and lack of potential to simultaneously edit multiple loci. Recently, prime editing (PE) has emerged as a new gene editing technology with the potential to overcome the above-mentioned limitations. Interestingly, PE not only has higher specificity but also does not require double-strand breaks. In addition, a minimum possibility of potential off-target mutant sites makes PE a preferred choice for therapeutic gene editing. Furthermore, PE has the potential to introduce insertion and deletions of all 12 single-base mutations at target sequences. Considering its potential, PE has been applied as a treatment option for genetic diseases including hemoglobinopathies. β-Thalassemia, for example, one of the most significant blood disorders characterized by reduced levels of functional hemoglobin, could potentially be treated using PE. Therapeutic reactivation of the γ-globin gene in adult β-thalassemia patients through PE technology is considered a promising therapeutic strategy. The current review aims to briefly discuss the genome editing strategies and potential applications of PE for the treatment of β-thalassemia. In addition, the review will also focus on challenges associated with the use of PE.

Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3' PAM-distal region, but not in the 5' PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3' PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.

The autobiographical genre is a problem when it is considered inside the theatral genre. Because the dramatical author express himself through the mouth of fictional characters, the words of the characters could not be considered as the expression of the author himself. However, it seems that through the dialogues that can be heard, the situations, the characters and the places that are staged, some plays, especially since the 18th century, which represents a major step in self-expression, contains the signs of an autobiographical expression. Although the autobiographical genre as defined by P. Lejeune or G. Gusdorf does not exists in the theatre genre, we will study here how it nevertheless expresses itself, that is to say almost always in a hidden, implicit, ambiguous, diverted, but probable way. We will think of a method allowing the reader, but also the member of the audience, to identify and testify the presence of this autobiographical expression by emphazising some textual and scenic aspects. We will also work on connecting the autobiographical expression with the relationship that live the authors of the new work contained in our corpus with the audience. This relationship being fully opend up in emotion. Some feelings, coming with physical reactions felt by the reader/audience, are created by the recognition of the author's voice in a drama that mixes autobiographical and fiction. We will take an interest in the autobiographical expression of the dramatical author through the perspective of his particular relationship with the audience, relationship that contrasts with Brecht's principles, which have ruled the theatrical creation in the 20th century.

Multiplexed Superresolution CRISPR Imaging of Chromatin in Living Cells

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used for genome editing in various fruit trees, including apple (Malus × domestica). In previous studies, transfer DNA (T-DNA) expressing genome editing tools, Streptococcus pyogenes Cas9 (SpCas9) and single guide RNA (sgRNA), was stably integrated into the apple genome via Agrobacterium-mediated transformation. However, due to self-incompatibility, long generation period, and the high heterozygosity of apple, removing only the integrated T-DNA from the apple genome by crossbreeding while maintaining the introduced varietal trait is difficult. Therefore, an efficient SpCas9-sgRNA delivery system without transgene insertion is required for genome editing of apple. In this study, we used geminivirus-derived replicons (GVRs) for the transient expression of genome editing tools. Small DNA vectors were deconstructed by splitting the elements necessary for the production of GVRs from bean yellow dwarf virus into two vectors. Production of GVRs using these vectors was demonstrated in Arabidopsis and apple cells. Genome editing was improved by using the GVR-producing vectors with genome editing tools in Arabidopsis protoplasts. The use of the GVR-producing vectors for SpCas9 and sgRNA delivery into apple leaves improved the expression levels of SpCas9 and sgRNA, enabling the detection of targeted mutations introduced in the endogenous apple genome. These findings demonstrate the utility of GVRs in genome editing via transient gene expression in apple. It can be expected that our GVR-based genome editing technology has potential utility for transgene-free genome editing in apple.

Base editors are powerful tools for making precise single-nucleotide changes in the genome. However, they can lead to unintended insertions and deletions at the target sites, which is a significant limitation for clinical applications. In this study, we aimed to eliminate unwanted indels at the target sites caused by various evolved base editors. Accordingly, we applied dead Cas9 instead of nickase Cas9 in the base editors to induce accurate substitutions without indels. Additionally, we tested the use of chromatin-modulating peptides in the base editors to improve nucleotide conversion efficiency. We found that using both dead Cas9 and chromatin-modulating peptides in base editing improved the nucleotide substitution efficiency without unintended indel mutations at the desired target sites in human cell lines and mouse primary myoblasts. Furthermore, the proposed scheme had fewer off-target effects than conventional base editors at the DNA level. These results indicate that the suggested approach is promising for the development of more accurate and safer base editing techniques for use in clinical applications.

CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.

Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from Prevotella and Francisella) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol, we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs. This protocol enables indel formation and homologous recombination–mediated precise editing at multiple loci. With the delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting and a MAFB-targeting crRNA array, efficient multiplex genome editing at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs could be achieved in a single experiment. The edited hPSCs expressed pluripotency markers and could differentiate into neurons in vitro. This system also generated INS reporter hPSCs with a 6 kb cassette knockin at the INS locus. The INS reporter cells can differentiate into β-cells that express tdTomato and luciferase, permitting fluorescence-activated cell sorting of hPSC-β-cells. By targeted screening of potential off-target sequences that are most homologous to crRNA sequences, no off-target mutations were detected in any of the tested sequences. This work provides an efficient and flexible system for precise genome editing in mammalian cells including hPSCs with the benefits of less off-target effects.Key features• A single-vector system to deliver Cpf1 and crRNA enables the sorting of transfected cells• Efficient and simultaneous multi-modular genome editing exemplified by mutation of MAFB and knockin of AAVS1 loci in a single experiment• Edited PSCs showed minimal off-target effects and can be differentiated into multiple cell types

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.

Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells

The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized the field of genetic engineering and molecular biology, offering unprecedented opportunities for precise genome editing. Initially discovered as a natural defense mechanism in bacteria, CRISPR-Cas9 technology enables targeted modifications of DNA, paving the way for significant advancements in gene therapy. This review article explores the multifaceted applications of CRISPR, focusing on its transformative potential in treating genetic diseases and various forms of cancer. We delve into the mechanisms by which CRISPR operates, emphasizing its dual-step process of targeting and cutting DNA, and highlighting its versatility in investigating gene functions and developing therapeutic strategies. Specific applications in genetic disorders, including sickle cell disease and cystic fibrosis, showcase the promising outcomes of ongoing pre-clinical and clinical trials. In the realm of oncology, CRISPR’s ability to edit oncogenes and enhance immune cell functions exemplifies its role in developing personalized cancer therapies. However, the rapid advancement of CRISPR technology also necessitates critical discussions around ethical considerations and regulatory challenges, particularly concerning off-target effects and the implications of germline editing. Looking ahead, innovations such as CRISPR 2.0, base editing, and prime editing are set to further expand the therapeutic landscape, enhancing the precision and safety of gene editing. This article underscores the importance of collaborative efforts across scientific, ethical, and regulatory domains to navigate the complexities of CRISPR technology while maximizing its benefits for patients and society.