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NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens.
The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for fundamental research and biotechnology. To fully harness the potential of this bacterium, highly efficient genome editing methods are indispensable to create strains tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This highly efficient natural transformation is able to mediate uptake of multiple DNA fragments, thereby allowing for multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR-Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR-Cas9 targeting the wild type sequence, and thus leading to death of non-edited cells. Through cell killing with efficiencies of up to 99.999%, integration of antibiotic resistance markers became dispensable, enabling scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100%. Further, we confirmed its applicability for simultaneous deletion of multiple chromosomal regions. Lastly, we showed that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby broadens the target spectrum.
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Prime Editing for Human Gene Therapy: Where Are We Now?
Gene therapy holds tremendous potential in the treatment of inherited diseases. Unlike traditional medicines, which only treat the symptoms, gene therapy has the potential to cure the disease by addressing the root of the problem: genetic mutations. The discovery of CRISPR/Cas9 in 2012 paved the way for the development of those therapies. Improvement of this system led to the recent development of an outstanding technology called prime editing. This system can introduce targeted insertions, deletions, and all 12 possible base-to-base conversions in the human genome. Since the first publication on prime editing in 2019, groups all around the world have worked on this promising technology to develop a treatment for genetic diseases. To date, prime editing has been attempted in preclinical studies for liver, eye, skin, muscular, and neurodegenerative hereditary diseases, in addition to cystic fibrosis, beta-thalassemia, X-linked severe combined immunodeficiency, and cancer. In this review, we portrayed where we are now on prime editing for human gene therapy and outlined the best strategies for correcting pathogenic mutations by prime editing.
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Recent Research Trends in Stem Cells Using CRISPR/Cas-Based Genome Editing Methods.
The clustered regularly interspaced short palindromic repeats (CRISPR) system, a rapidly advancing genome editing technology, allows DNA alterations into the genome of organisms. Gene editing using the CRISPR system enables more precise and diverse editing, such as single nucleotide conversion, precise knock-in of target sequences or genes, chromosomal rearrangement, or gene disruption by simple cutting. Moreover, CRISPR systems comprising transcriptional activators/repressors can be used for epigenetic regulation without DNA damage. Stem cell DNA engineering based on gene editing tools has enormous potential to provide clues regarding the pathogenesis of diseases and to study the mechanisms and treatments of incurable diseases. Here, we review the latest trends in stem cell research using various CRISPR/Cas technologies and discuss their future prospects in treating various diseases.
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Innovations in CRISPR-Based Therapies.
Gene and cell therapies have shown tremendous advancement in the last 5 years. Prominent examples include the successful use of CRISPR-edited stem cells for treating blood disorders like sickle cell anemia and beta-thalassemia, and ongoing clinical trials for treating blindness. This mini-review assesses the status of CRISPR-based therapies, both in vivo and ex vivo, and the challenges associated with clinical translation. In vivo CRISPR therapies have been used to treat eye and liver diseases due to the practicality of delivering editing components to the target tissue. In contrast, even though ex vivo CRISPR therapy involves cell isolation, expansion, and infusion, its advantages include characterizing the gene edits before infusion and restricting off-target effects in other tissues. Further, the safety, affordability, and feasibility of CRISPR therapies, especially for treating large number of patients, are discussed.
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CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells.
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Cytosine base editors go off-target.
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Plant gene editing through de novo induction of meristems.
Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing.
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UGI relocation inside Cas9 reduces Cas9 dependent off target effects in cytosine base editors.
Cytosine base editors (CBEs) achieve precise C-to-T conversions by addition of uracil DNA glycosylase inhibitor (UGI) with Cas9 nickase (nCas9) and cytidine deaminase, and the conventional fusion at the nCas9 carboxyl terminus effectively inhibits uracil excision repair to enhance editing efficiency. However, despite potent on-target activity, classical CBEs exhibit significant Cas9-dependent DNA off-target effects that necessitate optimization for future applications. Here we present a strategic UGI relocation through internal fusion within the nCas9 architecture. This spatial reorganization maintains comparable on-target editing efficiency while substantially reducing Cas9-dependent DNA off-target activity. Our findings establish an alternative engineering paradigm to develop high-fidelity CBEs, offering an improved platform for widespread genome editing applications.
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One Versatile Cas9-Integrated Single-Tube Duplex Quantitative Real-Time PCR System for Rapid Analysis of CRISPR/Cas-Induced Mutants.
Clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 gene editing has become a common tool for rapid crop and animal breeding, but efficiently screening out and genotyping for the CRISPR/Cas9-induced mutant lines at a low cost remains challenging. Using rice (Oryza sativa L.) samples genetically edited at the Waxy locus as an example, we developed a single-tube duplex quantitative real-time PCR assisted by an in vitro CRISPR/Cas9 cleavage (Cc-qPCR) method to screen for expected genetically edited lines, identify genotypes, and evaluate gene-editing frequency. In Cc-qPCR, genomic DNA is first cleaved at the target site by the single-guide RNA (sgRNA)/Cas9 complex and then quantified with qPCR to assess for the presence of a mutant and identify sample genotypes. Our findings suggest that Cc-qPCR can successfully identify mutants with small insertions or deletions (indels), even in mutant lines with single-base indels or substitutions. Cc-qPCR was also able to successfully identify heterozygous and homozygous mutants. The sensitivity of Cc-qPCR was determined to be as low as 0.5%, indicating that the method could be used to evaluate the editing efficiency of gene-editing systems. After testing our novel method on Waxy locus-edited rice offspring, our results show that Cc-qPCR is an accurate and effective approach to rapidly identify expected mutants and their genotypes and to evaluate editing efficiency. This method will prove useful for increasing the efficiency and range of molecular breeding techniques.
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Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection.
Although highly active antiretroviral therapy (HAART) can robustly control human immunodeficiency virus (HIV) infection, the existence of latent HIV in a form of proviral DNA integrated into the host genome makes the virus insensitive to HAART. This requires patients to adhere to HAART for a lifetime, often leading to drug toxicity or viral resistance to therapy. Current genome-editing technologies offer different strategies to reduce the latent HIV reservoir in the body. In this review, we systematize the research on CRISPR/Cas-based anti-HIV therapeutic methods, discuss problems related to viral escape and gene editing, and try to focus on the technologies that effectively and precisely introduce genetic modifications and confer strong resistance to HIV infection. Particularly, knock-in (KI) approaches, such as mature B cells engineered to produce broadly neutralizing antibodies, T cells expressing fusion inhibitory peptides in the context of inactivated viral coreceptors, or provirus excision using base editors, look very promising. Current and future advancements in the precision of CRISPR/Cas editing and its delivery will help extend its applicability to clinical HIV therapy.
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