As the most effective and popular gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR) technology has produced a revolution in biological fundamental research, medicine and biotechnology. In this review, we describe the history of the CRISPR-CRISPR-associated protein (Cas) systems, the tools of CRISPR-Cas9, CRISPR-FnCas9/RCas9, CRISPR-Cas13 and CRISPR-Cas12a, and then some comments we need to think about.
Hereditary retinopathy (IRD) is a group of congenital retinal degenerative diseases caused by lesions in photoreceptor cells or retinal pigment epithelial cells. It has a high degree of genetic and clinical heterogeneity and is an important cause of incurable blindness in adolescents. Splicing variation is a special type of genetic variation caused by gene sequence mutations interfering with the normal RNA splicing process. It leads to abnormal rearrangement patterns of messenger ribonucleic acid during maturation, which is essentially different from the physiological isomers produced by normal selective splicing. IRD splicing variations mainly include classical site variations and deep intron site variations. Many breakthroughs have been made in targeted therapy for these variations. Antisense oligonucleotides, clustered regularly interspaced short palindromic repeats and associated protein 9 binding induced pluripotent stem cells, small molecule compounds, and spliceosome-mediated RNA trans-splicing techniques all show good application prospects. However, at present, related research is still in a fragmented state. A systematic review of splicing mutations and related treatment progress will provide important support for in-depth clarification of their pathogenic mechanisms and the development of effective therapies, bringing a glimmer of hope to such diseases that were once regarded as "incurable". Although there are still challenges such as low detection efficiency, poor treatment safety and individualized differences at present, with the continuous progress of molecular biology and gene therapy technology, it is gradually becoming possible to achieve precise diagnosis and radical individualized treatment of splicing mutation-related IRD, bringing hope to patients.
Objective To investigate the causes and management strategies for lower limb ischemic necrosis following xenogeneic heterotopic heart transplantation from a multigene-edited pig to a rhesus monkey. Methods A xenogeneic heterotopic heart transplantation was performed on December 16, 2023, at the Institute of Experimental Animals of Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, using a quintuple-gene-edited pig as the donor and a rhesus monkey as the recipient. On postoperative day (POD) 9, the recipient monkey underwent left lower limb amputation due to ischemic necrosis. Blood samples were collected at various time points after transplantation for analysis of hematologic parameters, liver and renal function, myocardial enzymes, and coagulation profiles. Ultrasound and computed tomography (CT) were used to evaluate anastomotic patency and cardiac structure. Immunological assays, including complement-dependent cytotoxicity (CDC) and IgG/IgM antibody detection, combined with clinical observations, were employed to assess rejection type and therapeutic response. Results The recipient monkey survived for 46 days after transplantation. Echocardiography demonstrated preserved biventricular systolic function in the recipient’s native heart, with left ventricular ejection fraction (LVEF) consistently exceeding 50%. In the donor pig heart, left ventricular endocardial thickening was noted on POD 9, followed by right ventricular endocardial thickening on POD 24, while LVEF remained around 35%. No hyperacute or acute rejection was detected immunologically. CDC positivity ranged between 3.4% and 5.1%, with IgG/IgM antibody binding trends consistent with CDC results. Following amputation, the recipient exhibited elevated inflammatory markers, coagulopathy, and reactive thrombocytosis, which later normalized. Immunohistochemical staining of the necrotic limb revealed arterial and venous thrombosis; however, no T-cell or B-cell infiltration was observed in vascular structures, thrombi, nerves, muscles, fascia, or skin tissues, with CD3 and CD20 staining both negative. Conclusion Limb ischemia after xenogeneic heart transplantation may be associated with lower extremity vascular thrombosis triggered by local trauma in the context of transplantation-induced inflammatory activation and coagulation dysfunction. While no clear lymphocyte-mediated rejection was observed, further studies are needed to explore the potential role of non-lymphocyte-mediated immune mechanisms.
