RESUMO
The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.
Assuntos
Sistemas CRISPR-Cas , Dependovirus , Edição de Genes , Vetores Genéticos , Dependovirus/genética , Humanos , Edição de Genes/métodos , Vetores Genéticos/genética , Animais , Células HEK293 , Terapia Genética/métodos , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Camundongos , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
OBJECTIVE: To compare the contraceptive efficacy, tolerability and bleeding patterns of Implanon and Norplant. METHODS: One hundred healthy female volunteers received either Implanon (75 cases) or Norplant (25 cases) for 4 years, followed up with complain, bleeding patterns, hemoglobin, blood pressure, weight. Record the time of insert and remove. RESULTS: There were no pregnancies during the study. Per 90-day reference period, the average number of bleeding-spotting days with Implanon decreased from 33 day in the first period to 21 day in the last year, and Norplant decreased from 31 day to 20 day. The average bleeding number of times was 2.25 of Implanon and 2.99 of Norplant per reference period (P < 0.05). The mean insert time was 11 second of Implanon and 103 second of Norplant. The mean remove time was 27 second of Implanon and 102 second of Norplant (P < 0.001). CONCLUSION: Both contraceptive systems demonstrated excellent contraceptive efficacy and were well tolerated. Because of its single-rod design, Implanon was significantly quicker to insert and remove than the multiple-capsule system.