Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

Investigating Temperature-Dependent Microscopic Deformation in Tough and Self-Healing Hydrogel Using Time-Resolved USAXS.

Tough and self-healing hydrogels are typically sensitive to loading rates or temperatures due to the dynamic nature of noncovalent bonds. Understanding the structure evolution under varying loading conditions can provide valuable insights for developing new tough soft materials. In this study, polyampholyte (PA) hydrogel with a hierarchical structure is used as a model system. The evolution of the microscopic structure during loading is investigated under varied loading temperatures. By combining ultra-small angle X-ray scattering (USAXS) and Mooney-Rivlin analysis, it is elucidated that the deformation of bicontinuous hard/soft phase networks is closely correlated with the relaxation dynamics or strength of noncovalent bonds. At high loading temperatures, the gel is soft and ductile, and large affine deformation of the phase-separated networks is observed, correlated with the fast relaxation dynamics of noncovalent bonds. At low loading temperatures, the gel is stiff, and nonaffine deformation occurs from the onset of loading due to the substantial breaking of noncovalent bonds and limited chain mobility as well as weak adaptation of phase deformation to external stretch. This work provides an in-depth understanding of the relationship between structure and performance of tough and self-healing hydrogels.

Computational design of an amidase by combining the best electrostatic features of two promiscuous hydrolases.

While there has been emerging interest in designing new enzymes to solve practical challenges, computer-based options to redesign catalytically active proteins are rather limited. Here, a rational QM/MM molecular dynamics strategy based on combining the best electrostatic properties of enzymes with activity in a common reaction is presented. The computational protocol has been applied to the re-design of the protein scaffold of an existing promiscuous esterase from Bacillus subtilis Bs2 to enhance its secondary amidase activity. After the alignment of Bs2 with a non-homologous amidase Candida antarctica lipase B (CALB) within rotation quaternions, a relevant spatial aspartate residue of the latter was transferred to the former as a means to favor the electrostatics of transition state formation, where a clear separation of charges takes place. Deep computational insights, however, revealed a significant conformational change caused by the amino acid replacement, provoking a shift in the pK a of the inserted aspartate and counteracting the anticipated catalytic effect. This prediction was experimentally confirmed with a 1.3-fold increase in activity. The good agreement between theoretical and experimental results, as well as the linear correlation between the electrostatic properties and the activation energy barriers, suggest that the presented computational-based investigation can transform in an enzyme engineering approach.