Advancing gene and base editing for cardiovascular disease: overcoming barriers in delivery, precision and safety.

CRISPR Genome Editing: Into the Second Decade

Toward CRISPR Therapies for Cardiomyopathies.

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

Research progress of gene editing technology in neurological diseases.

The EHA Research Roadmap: Hematopoietic Stem Cell Gene Therapy.

Advances and challenges in adeno-associated viral inner-ear gene therapy for sensorineural hearing loss

Genetics has transformed from a purely descriptive science to a manipulative science. The first techniques for genome editing came in the mid-1980s in the studies of Oliver Smithies and Mario Capecchi but made huge advances through the discovery of the targeted endonucleases such as zinc finger endonuclease, transcription activator-like effectors (TALEs) and clustered regularly interspaced short palindromic repeats (CRISPR) and their associated families of scissors-like Cas endonucleases (CRISPR-associated proteins). It is an understatement to say that not only have these tools enabled targeted sequence editing of the genome but that they have in fact revolutionized most of biological science. Methods are rapidly being developed to deliver the CRISPR-Cas9 complexes to human cells and tissues, especially in vivo, and to do so safely, efficiently and non-immunogenically. The well-established methods for gene transfer, including physical methods of electroporation, microinjection, and hydrodynamic injection, the use of nonviral systems such as lipid nanoparticles and adenovirus, lentivirus and adeno-associated virus (AAV) viral vectors are all being adapted to the in vitro and in vivo delivery of CRISPR-Cas editing complexes. Clinical efficacy has been established by a recently approved and probably curative CRISPR-based therapy for sickle cell disease that promises to revolutionize treatment of that disease. For some disorders, a variation of genome editing involving editing at single base sites is being developed.

Cas-Clover Editing Efficiency and Off-Target Activity in Human Hepatocytes at the KLKB1 Locus

In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.

BackgroundThe CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a).ResultsWe created new AsCas12a variant termed “AsCas12a-Plus” with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity.ConclusionsOur discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

In recent years, gene editing technologies, especially CRISPR-Cas9, have become key tools for enhancing the effectiveness of immunotherapy and are widely applied in the treatment of cancer, viral infections, and genetic diseases. By improving the specificity and durability of immune cells, gene editing boosts the efficacy of anti-tumor therapies, such as CAR-T cell therapy, and optimizes immune checkpoint inhibitors to enhance immune response and inhibit cancer immune evasion. Moreover, CRISPR demonstrates significant potential in antiviral immunity, particularly achieving breakthroughs in targeting HIV and other viral replication mechanisms. Despite the promising outlook of gene editing technology in clinical applications, it still faces challenges such as off-target effects, ethical concerns, and technical limitations. Researchers are actively exploring new editing tools (e.g., Cas12, Cas13) and delivery systems (e.g., lipid nanoparticles, adeno-associated viruses) to improve editing accuracy and safety. With the integration of gene editing with multiple therapies, the advancement of personalized medicine, and improvements in delivery technology, gene editing is expected to achieve wider clinical application in the future, drive the progress of precision medicine, and offer innovative treatment strategies for hard-to-treat cancers and genetic diseases.

Lipid nanoparticle-mediated intracameral mRNA delivery facilitates gene expression and editing in the anterior chamber of the eye.

CRISPR/Cas12a, a promising gene editing technology, faces limitations due to its requirement for a thymine (T)‐rich protospacer adjacent motif (PAM). Despite the development of Cas12a variants with expanded PAM profiles, many genomic loci, especially those with guanine‐cytosine (GC)‐rich PAMs, have remained inaccessible. This study develops a small RNA toxin‐aided strategy to evolve ErCas12a for targeting GC‐rich PAMs, resulting in the creation of enhanced ErCas12a (enErCas12a). EnErCas12a demonstrates the ability to recognize GC‐rich PAMs and target five times more PAM sequences than the wild‐type ErCas12a. Furthermore, enErCas12a achieves efficient gene editing in both bacterial and mammalian cells at various sites with non‐canonical PAMs, including GC‐rich PAMs such as GCCC, CGCC, and GGCC, which are inaccessible to previous Cas12a variants. Moreover, enErCas12a effectively targets PAM sequences with a GC content exceeding 75% in mammalian cells, providing a valuable alternative to the existing Cas12a toolkit. Importantly, enErCas12a maintains high specificity at targets with canonical PAMs, while also demonstrating enhanced specificity at targets with non‐canonical PAMs. Collectively, this work establishes enErCas12a as a promising tool for gene editing in both eukaryotes and prokaryotes.

There are a lot of diseases known today, which are caused by genetic abnormalities. Advances in genetics and biotechnology brought about gene editing technologies that can produce almost any gene, which ultimately led to the emergence of a new class of medicines - gene therapy products (GTPs). The aim of the study was to analyse international experience in development and authorisation of GTPs. The review highlights the challenges in GTP development, related to the search for an optimal approach to therapeutic gene delivery to the target cells. Viral vectors were shown to be a promising gene delivery system, with adenovirus (AV) and adeno-associated virus (AAV) based products demonstrating the highest efficacy and safety. The paper reviews current approaches to gene editing that allow modification of AVs and AAVs to improve GTP efficacy and safety. These modifications are carried out with the aim of, e.g., including a large therapeutic gene into a viral vector, decreasing viral protein expression levels, and decreasing viral vector immunogenicity. The review summarises GTP authorisation procedures in the USA and the European Union, including data on FDA and EMA subcommittees and departments entrusted with advisory functions. The paper mentions that there is one Russian-produced GTP authorised in the Russian Federation, and some other GTPs are in the pipeline. Therefore, the Russian regulatory framework and the Eurasian regulations and recommendations should be updated in order to accommodate for GTP development and authorisation.

Gene editing offers dietary benefits