Development of miniature base editors using engineered IscB nickase.

As a miniature RNA-guided endonuclease, IscB is presumed to be the ancestor of Cas9 and to share similar functions. IscB is less than half the size of Cas9 and thus more suitable for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells limits its in vivo applications. Here we describe the engineering of OgeuIscB and its corresponding ωRNA to develop an IscB system that is highly efficient in mammalian systems, named enIscB. By fusing enIscB with T5 exonuclease (T5E), we found enIscB-T5E exhibited comparable targeting efficiency to SpG Cas9 while showing reduced chromosome translocation effects in human cells. Furthermore, by fusing cytosine or adenosine deaminase with enIscB nickase, we generated miniature IscB-derived base editors (miBEs), exhibiting robust editing efficiency (up to 92%) to induce DNA base conversions. Overall, our work establishes enIscB-T5E and miBEs as versatile tools for genome editing.

Engineered IscB-ωRNA system with expanded target range for base editing.

As the evolutionary ancestor of Cas9 nuclease, IscB proteins serve as compact RNA-guided DNA endonucleases and nickases, making them strong candidates for base editing. Nevertheless, the narrow targeting scope limits the application of IscB systems; thus, it is necessary to find more IscBs that recognize different target-adjacent motifs (TAMs). Here, we identified 10 of 19 uncharacterized IscB proteins from uncultured microbes with activity in mammalian cells. Through protein and ωRNA engineering, we further enhanced the activity of IscB ortholog IscB.m16 and expanded its TAM scope from MRNRAA to NNNGNA, resulting in a variant named IscB.m16*. By fusing the deaminase domains with IscB.m16* nickase, we generated IscB.m16*-derived base editors that exhibited robust base-editing efficiency in mammalian cells and effectively restored Duchenne muscular dystrophy proteins in diseased mice through single adeno-associated virus delivery. Thus, this study establishes a set of compact base-editing tools for basic research and therapeutic applications.

High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects.

CRISPR-Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.

Assessment of benthic invertebrate diversity and river ecological status along an urbanized gradient using environmental DNA metabarcoding and a traditional survey method.

Benthic invertebrate diversity is one of the most commonly used bioindicators for assessing aquatic ecosystem health in river systems. Although an increasing number of studies have focused on assessing benthic invertebrate diversity using environmental DNA metabarcoding and traditional survey methods, benthic invertebrate diversity and ecological status assessments performed across different landscapes within river systems have not been well documented. Here, the diversity and ecological status of benthic invertebrates and the influence of water quality on the invertebrate assemblage distribution along an urbanization gradient in rivers from the Jingjinji (JJJ) region, China, were investigated using eDNA metabarcoding and the traditional method. With the combination of the two methods, 395 benthic invertebrates from 6 phyla, 27 orders, 94 families, and 222 genera were identified. The species richness of the benthic invertebrate community in the mountain area was significantly higher than that in the urban and agricultural areas. Compared to the traditional results, eDNA metabarcoding obtained a significantly greater number of species from every sampling site (P = 0.000) and detected a notably higher abundance in Annelida (P = 0.000). Furthermore, the nonmetric multidimensional scaling (NMDS) and permutational multivariate analysis of variance (PERMANOVA) based on the Bray-Curtis dissimilarity index indicated that the benthic invertebrate communities from the different habitats were discriminated more accurately and easily using eDNA metabarcoding (P = 0.038) than with the traditional method (P = 0.829). Additionally, the assemblages identified by eDNA metabarcoding were more closely linked to water quality and could be realistically used to assess the ecological status of rivers. Our findings highlight that eDNA metabarcoding could represent a rapid and reliable method for estimating benthic invertebrate diversity and ecological status in river systems.