All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Knockout and Replacement Gene Surgery to Treat Rhodopsin-Mediated Autosomal Dominant Retinitis Pigmentosa.

Mutations in the rhodopsin (RHO) gene are the predominant causes of autosomal dominant retinitis pigmentosa (adRP). Given the diverse gain-of-function mutations, therapeutic strategies targeting specific sequences face significant challenges. Here, we provide a universal approach to conquer this problem: we have devised a CRISPR-Cas12i-based, mutation-independent gene knockout and replacement compound therapy carried by a dual AAV2/8 system. In this study, we successfully delayed the progression of retinal degeneration in the classic mouse disease model RhoP23H, and also RhoP347S, a new native mouse mutation model we developed. Our research expands the horizon of potential options for future treatments of RHO-mediated adRP.

Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12iMax.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12iMax, a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12iMax in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163, and MSTN via Cas12iMax in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12iMax for gene editing in livestock animals and demonstrated the potential application of Cas12iMax in the field of animal trait improvement for agricultural production.

High-efficiency targeted transgene integration via primed micro-homologues.

Due to the difficulties in precisely manipulating DNA repair pathways, high-fidelity targeted integration of large transgenes triggered by double-strand breaks is inherently inefficient. Here, we exploit prime editors to devise a robust knock-in (KI) strategy named primed micro-homologues-assisted integration (PAINT), which utilizes reverse-transcribed single-stranded micro-homologues to boost targeted KIs in different types of cells. The improved version of PAINT, designated PAINT 3.0, maximizes editing efficiency and minimizes off-target integration, especially in dealing with scarless in-frame KIs. Using PAINT 3.0, we target a reporter transgene into housekeeping genes with editing efficiencies up to 80%, more than 10-fold higher than the traditional homology-directed repair method. Moreover, the use of PAINT 3.0 to insert a 2.5-kb transgene achieves up to 85% KI frequency at several therapeutically relevant genomic loci, suggesting its potential for clinical applications. Finally, PAINT 3.0 enables high-efficiency non-viral genome targeting in primary T cells and produces functional CAR-T cells with specific tumor-killing ability. Thus, we establish that the PAINT method is a powerful gene editing tool for large transgene integrations and may open new avenues for cell and gene therapies and genome writing technologies.

CDetection.v2: One-pot assay for the detection of SARS-CoV-2.

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing.

The naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools. However, the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells, which significantly limits their utility. Here, we have developed a method termed Improving Editing Activity by Synergistic Engineering (MIDAS). This method exerts a synergistic effect to improve mammalian genome-editing efficiency of a wide range of CRISPR-Cas systems by enhancing the interactions between Cas nuclease with the protospacer adjacent motif (PAM) and the single-stranded DNA (ssDNA) substrate in the catalytic pocket simultaneously. MIDAS robustly and significantly increased the gene-editing efficiency of Cas12i, Cas12b, and CasX in human cells. Notably, a Cas12i variant, Cas12i Max , exhibited robust activity with a very broad PAM range (NTNN, NNTN, NAAN, and NCAN) and higher efficiency than the current widely used Cas nucleases. A high-fidelity version of Cas12i Max (Cas12i HiFi ) has been further engineered to minimize off-target effects. Our work provides an expandable and efficacious method for engineering Cas nucleases for robust mammalian genome editing.

Gene editing and its applications in biomedicine.

The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.