An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

The ScCas9++ variant expands the CRISPR toolbox for genome editing in plants.

The development of clustered regularly interspaced palindromic repeats (CRISPR)-associated protein (Cas) variants with a broader recognition scope is critical for further improvement of CRISPR/Cas systems. The original Cas9 protein from Streptococcus canis (ScCas9) can recognize simple NNG-protospacer adjacent motif (PAM) targets, and therefore possesses a broader range relative to current CRISPR/Cas systems, but its editing efficiency is low in plants. Evolved ScCas9+ and ScCas9++ variants have been shown to possess higher editing efficiencies in human cells, but their activities in plants are currently unknown. Here, we utilized codon-optimized ScCas9, ScCas9+ and ScCas9++ and a nickase variant ScCas9n++ to systematically investigate genome cleavage activity and cytidine base editing efficiency in rice (Oryza sativa L.). This analysis revealed that ScCas9++ has higher editing efficiency than ScCas9 and ScCas9+ in rice. Furthermore, we fused the evolved cytidine deaminase PmCDA1 with ScCas9n++ to generate a new evoBE4max-type cytidine base editor, termed PevoCDA1-ScCas9n++ . This base editor achieved stable and efficient multiplex-site base editing at NNG-PAM sites with wider editing windows (C- 1 -C17 ) and without target sequence context preference. Multiplex-site base editing of the rice genes OsWx (three targets) and OsEui1 (two targets) achieved simultaneous editing and produced new rice germplasm. Taken together, these results demonstrate that ScCas9++ represents a crucial new tool for improving plant editing.

A chromosome-level genome assembly of the wild rice Oryza rufipogon facilitates tracing the origins of Asian cultivated rice.

Oryza rufipogon Griff. is a wild progenitor of the Asian cultivated rice Oryza sativa. To better understand the genomic diversity of the wild rice, high-quality reference genomes of O. rufipogon populations are needed, which also facilitate utilization of the wild genetic resources in rice breeding. In this study, we generated a chromosome-level genome assembly of O. rufipogon using a combination of short-read sequencing, single-molecule sequencing, BioNano and Hi-C platforms. The genome sequence (399.8 Mb) was assembled into 46 scaffolds on the 12 chromosomes, with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb, respectively. The genome contains 36,520 protein-coding genes, and 49.37% of the genome consists of repetitive elements. The genome has strong synteny with those of the O. sativa subspecies indica and japonica, but containing some large structural variations. Evolutionary analysis unveiled the polyphyletic origins of O. sativa, in which the japonica and indica genome formations involved different divergent O. rufipogon (including O. nivara) lineages, accompanied by introgression of genomic regions between japonica and indica. This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O. sativa subspecies, and valuable information for basic research and rice breeding.

An interactive method for achieving ergonomically optimum conditions during laparoscopic surgery.

Laparoscopy is widely used during living kidney donation, nephrectomy, bariatric surgery, and surgery for gastrointestinal tumors and colorectal cancer. However, laparoscopic surgery requires prolonged use of instruments and has low mechanical efficiency. In addition, to meet specific surgical demands while visualizing the surgical area on the screen, surgical personnel frequently violate the postures proposed by human factor engineering; this naturally results in a physical burden on the personnel. In this study, using laparoscopic nephrectomy as an example, an auxiliary hoisting device for surgery was designed, and pedals from a variety of equipment were integrated into the auxiliary plant. Both entity testing conducted in the hospital and 3D surgical simulation of the auxiliary plant showed that this device could improve compliance with human factor engineering recommendations during laparoscopic surgery and could also promote interaction and tacit understanding between surgical personnel, thereby providing ergonomically optimum conditions during laparoscopy.

Application of the laser Doppler flowmeter for measurement of blood pressure and functional parameters of microcirculation.

The laser Doppler flowmeter has become an effective tool for the clinical study of microcirculation with non-invasive measurements and excellent velocity resolution. Microcirculation flow measurements cannot be used as reference criteria for circulation function. Thus, the relative change in fingertip microcirculation was measured through inflation and deflation of an automatic sphygmomanometer wrapped around the arm, and the blood pressure and functional parameters of circulation, such as biological zero (BZ), peak flow (PF) and time to peak flow (tp), were determined. For 50 healthy participants, the correlation coefficients for the laser Doppler flowmeter and oscillometric results were 0.950 and 0.838 for systolic and diastolic pressure, respectively. The mean and standard errors for both methods fell within the range specified in the Association for the Advancement of Medical Instrumentation standards. The BZ of an edema patient was about 5 times higher than normal. For patients with peripheral arterial occlusive disease (PAOD), the PF could not be determined, and the tp was about twice the normal value. The accuracy of blood pressure measurements using the laser Doppler flowmeter was comparable to that of the commonly used oscillometric sphygmomanometer, and the physiological circulation functional parameters were useful in identifying signs of edema and PAOD.

A novel dynamic sensing of wearable digital textile sensor with body motion analysis.

This work proposes an innovative textile sensor system to monitor dynamic body movement and human posture by attaching wearable digital sensors to analyze body motion. The proposed system can display and analyze signals when individuals are walking, running, veering around, walking up and down stairs, as well as falling down with a wearable monitoring system, which reacts to the coordination between the body and feet. Several digital sensor designs are embedded in clothing and wear apparel. Any pressure point can determine which activity is underway. Importantly, wearable digital sensors and a wearable monitoring system allow adaptive, real-time postures, real time velocity, acceleration, non-invasive, transmission healthcare, and point of care (POC) for home and non-clinical environments.