Yanfei Zou

CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.

The eukaryotic genome is divided into chromosomal domains of distinct gene activities. Transcriptionally silent chromatin tends to encroach upon active chromatin. Barrier elements that can block the spread of silent chromatin have been documented, but the mechanisms of their function are not resolved. We show that the prokaryotic LexA protein can function as a barrier to the propagation of transcriptionally silent chromatin in yeast. The barrier function of LexA correlates with its ability to disrupt local chromatin structure. In accord with this, (CCGNN)(n) and poly(dA-dT), both of which do not favor nucleosome formation, can also act as efficient boundaries of silent chromatin. Moreover, we show that a Rap1p-binding barrier element also disrupts chromatin structure. These results demonstrate that nucleosome exclusion is one of the mechanisms for the establishment of boundaries of silent chromatin domains.

Phosphor‐converted near‐infrared (NIR) LEDs are becoming increasingly demanded as miniature, portability, and broad emission spectrum. In this work, a class of Fe 3+ ‐activated double perovskite structured is reported ALaBB′O 6 (A = Ba, Sr, Ca; B–B′ = Li–Te, Mg–Sb) phosphors. Through the co‐substitution strategy at the A‐site and B‐B' sites, the emission spectral intensity and position of Fe 3+ ions can be tuned. Finally, by utilizing Ca 2+ at the A‐site and Mg–Sb co‐substitution for Li–Te, long‐wave NIR emission centered at 995 nm in CaLaMgSbO 6 : 0.6%Fe 3+ with a full width at half maximum of 147 nm and internal quantum efficiency of 54.05% is achieved. The effects of the double perovskite crystal structure on Fe 3+ photoluminescence properties are comprehensively analyzed. NIR LEDs are fabricated by encapsulating UV chips with the synthetic CaLaMgSbO 6 : 0.6%Fe 3+ phosphors, and their application value in night vision, nondestructive biological monitoring, and NIR detection is evaluated.

Due to the increasing need for data sharing in the age of big data, how to achieve data access control and implement user permission revocation in the blockchain environment becomes an urgent problem. To solve the above problems, we propose a novel blockchain-based data sharing scheme (BDSS) with fine-grained access control and permission revocation in this paper, which regards the medical environment as the application scenario. In this scheme, we separate the public part and private part of the electronic medical record (EMR). Then, we use symmetric searchable encryption (SSE) technology to encrypt these two parts separately, and use attribute-based encryption (ABE) technology to encrypt symmetric keys which used in SSE technology separately. This guarantees better fine-grained access control and makes patients to share data at ease. In addition, we design a mechanism for EMR permission grant and revocation so that hospital can verify attribute set to determine whether to grant and revoke access permission through blockchain, so it is no longer necessary for ciphertext re-encryption and key update. Finally, security analysis, security proof and performance evaluation demonstrate that the proposed scheme is safe and effective in practical applications.