抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

/pdf/genome-engineering-using-the-crispr-cas9-system-4x8nw03mgu.pdf

Genome engineering using the CRISPR-Cas9 system

The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

The new frontier of genome engineering with CRISPR-Cas9

http://knockout.cwru.edu/publications/hsucrisprreview.pdf

Development and applications of CRISPR-Cas9 for genome engineering.

https://limlab.ucsf.edu/papers/pdfs/lsq_2013.pdf

Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.

Base editors function in mouse fetuses and in the livers of adult mice to treat a genetic disorder.

Towards therapeutic base editing

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP(+)/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

/pdf/production-of-mutated-porcine-embryos-using-zinc-finger-66r7pb83as.pdf

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

BACKGROUND Volar plate avulsion fracture of the proximal interphalangeal (PIP) joint is one of the most common hand injuries In this study, we divided patients into two groups: patients with pure volar plate avulsion fracture, and patients with volar plate avulsion fracture concomitant with collateral ligament rupture The purpose of this study was to compare long-term surgical outcomes between the two groups As a secondary measure, the Mitek bone anchoring and polydioxanone (PDS) bone suturing techniques were compared METHODS A single-institutional retrospective review of the surgical treatment of volar plate avulsion fracture was performed The cases were divided into those with pure volar plate avulsion fracture (group A, n=15) and those with volar plate avulsion fracture concomitant with collateral ligament rupture (group B, n=15) Both groups underwent volar plate reattachment using Mitek bone anchoring or PDS bone suturing followed by 2 weeks of immobilization in a dorsal protective splint RESULTS The average range of motion of the PIP joint and extension lag were significantly more favorable in group A (P<005) Differences in age; follow-up period; flexion function; visual analog scale scores; disabilities of the arm, shoulder, and hand scores; and the grip strength ratio between the two groups were non-significant No significant differences were found in the surgical outcomes of Mitek bone anchoring and PDS bone suturing in group A CONCLUSIONS Overall, the surgical outcomes of volar plate reattachment were successful irrespective of whether the collateral ligaments were torn However, greater extension lag was observed in cases of collateral ligament injury

/pdf/volar-plate-avulsion-fracture-alone-or-concomitant-with-1k00ttqtjm.pdf

Volar plate avulsion fracture alone or concomitant with collateral ligament rupture of the proximal interphalangeal joint: A comparison of surgical outcomes.

Objectives: In this comparative analytic study the standard competency of American Dental Hygienists proposed by the America Dental Education Association was examined and compared with the competency of American school of entry-level to identify the competence required by domestic dental hygienists. Methods: Based on the standard competency presented by the America Dental Education Association the 109 schools which provide respective dental hygiene competency among 336 universities and colleges belonging to the entry-level were compared with each other, and the collected data were processed by SPSS 21.0. Results: The descriptive statistics upon overall competence were prepared and the results of survey revealed the highest average score of 7.53 for the Core competency. It was identified that there were statistically significant difference between two groups of the above(the top 25%) and below the average level(the 25% from the bottom) in all the competency. The competency of participation in local community and patients’ care were appeared as significant variables affecting the core competency of dental hygienists with the 76.4% of explanatory power, and the model reveals the statistically significant results(p<0.001). Conclusions: Based on these results it was identified that the ethical, communication skill, self-development efforts, and capability of critical thinking and judgment were necessary competency for the dental hygienists. Further efforts to integrate and standardize the competency of domestic dental hygienists are thus needed and based on these integrated and standardized competencies the integrated curricula to cultivate domestic dental hygienists should be developed.

https://www.koreascience.or.kr:443/article/JAKO201521159149428.pdf

A comparative analysis of competencies in American Dental Education Association and American Dental Hygiene Schools

Since its discovery, the CRISPR/Cas9 system has been at the focus of fundamental researchers, genome engineers, and the general public alike. Despite being in the spotlight for several years, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. We use single-molecule Foerster resonance energy transfer (smFRET) to reveal Cas9 target search mechanism with nanometer sensitivity. We have developed single-molecule assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the protospacer adjacent motif (PAM) sequence weakly, yet probing neighboring sequences via lateral diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with 3-dimensional diffusion and thus might aid PAM finding and consequently on-target binding.

https://www.biorxiv.org/content/biorxiv/early/2018/02/13/264879.full.pdf

Jin-Soo Kim

Papers

Towards therapeutic base editing

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

Volar plate avulsion fracture alone or concomitant with collateral ligament rupture of the proximal interphalangeal joint: A comparison of surgical outcomes.

A comparative analysis of competencies in American Dental Education Association and American Dental Hygiene Schools

CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion