Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system.

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.

Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair.

An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform.

The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-CoV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of 10-17 M (approximately 6 copies/µL). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of 10-16 M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.

[One-year combination therapy de novo of adefovir dipivoxil and lamivudine for decompensated cirrhosis related to HBV].

OBJECTIVE: To evaluate the efficacy and potential renal impairment of one-year combination therapy de novo of adefovir dipivoxil (ADV) and lamivudine (LMV) for decompensated cirrhosis related to HBV. METHODS: A total of 36 patients with decompensated cirrhosis related to HBV, nobody had nucleos(t)ide analogs (NAs) treatment history, were recruited and were divided into two group (control group and observation group) randomly. A monotherapy of LMV (100 mg per day) was selected to individuals in control group (n = 18), in contrast, a combination therapy de novo of ADV (10 mg per day) and LMV (100 mg per day) was applied to those in observation group (n = 18). Basic approaches including liver protection, symptom-driven intervention, and supporting therapy, were given to all of the individuals. A course of one year was applied to all. Liver function, Child-Pugh score, serum creatinine (sCr) level, virological response (VR) rate, and virological breakthrough rate were observed pro- and post- treatment, differences between the two populations were analysed statistically. RESULTS: (1) The averages of gender, age, HBeAg status, HBV viral load, sCr level, and Child-Pugh score were all compatible in the two groups at baseline (P > 0.05 for all). (2) At the endpoint of treatment, none of deaths was reported. Comparing with the status before treatment in each group itself, liver function, Child-Pugh score, and viral load were improved statistically (P < 0.01 for all), especially in observed group (P < 0.01 for all variables, vs control group), as for VR rate, result is significant superior to that of control group too (88.89% vs 66.67% , P < 0.05). (3) Virological breakthrough occurred to none in observed group and three cases (16.67%) in control group, all of them were confirmed to be rtM204V variant in the following detection of direct sequencing. (4) Elevated level of sCr didn't arised at the end of treatment in two groups. CONCLUSION: Present study reveals that in populations with decompensated cirrhosis related to HBV, one-year combination therapy de novo of ADV and LMV is superior to monotherapy of LMV, and the renal safety is favorable within one year.