Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining.

In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects.

Effect of infrahepatic inferior vena cava partial clamping on central venous pressure and intraoperative blood loss during laparoscopic hepatectomy.

BACKGROUND: Infrahepatic inferior vena cava (IVC) clamping is considered to be an effective method to reduce central venous pressure (CVP) and intraoperative bleeding in liver resection. However, its efficacy and safety during laparoscopic hepatectomy (LH) remain unclear. We perform this retrospective study to evaluate its efficacy and safety during LH. METHODS: Consecutive patients scheduled for LH from September 2014 to August 2019 were retrospectively reviewed. The intraoperative parameters and postoperative outcomes were analyzed. RESULTS: All patients in the infrahepatic IVC clamping group were able to tolerate partial clamping of IVC. The CVP was significantly decreased after infrahepatic IVC clamping without hemodynamic instability (8.7 ± 1.4 cmH2O vs. 2.1 ± 1.3 cmH2O, P = 0.000). Infrahepatic IVC clamping did not significantly reduce total blood loss (287.3 ± 112.5 mL vs. 301.4 ± 127.6 mL, P = 0.133) and blood loss during parenchymal transection (273.2 ± 107.9 mL vs. 296.5 ± 118.1 mL, P = 0.618) compared with the non-clamping group. In subgroup analysis, total blood loss and blood loss during parenchymal transection were significantly reduced in patients with moderate to severe cirrhosis in the clamping group (363.6 ± 71.2 mL vs. 473.4 ± 95.6 mL, P = 0.021), (358.7 ± 70.9 mL vs. 466.9 ± 94.5 mL, P = 0.016), respectively. The complications and hospital stay were comparable. CONCLUSIONS: In conclusion, these data suggest that infrahepatic IVC clamping may be safe and effective.

H2AX facilitates classical non-homologous end joining at the expense of limited nucleotide loss at repair junctions.

Phosphorylated histone H2AX, termed 'γH2AX', mediates the chromatin response to DNA double strand breaks (DSBs) in mammalian cells. H2AX deficiency increases the numbers of unrepaired DSBs and translocations, which are partly associated with defects in non-homologous end joining (NHEJ) and contributing to genomic instability in cancer. However, the role of γH2AX in NHEJ of general DSBs has yet to be clearly defined. Here, we showed that despite little effect on overall NHEJ efficiency, H2AX deficiency causes a surprising bias towards accurate NHEJ and shorter deletions in NHEJ products. By analyzing CRISPR/Cas9-induced NHEJ and by using a new reporter for mutagenic NHEJ, we found that γH2AX, along with its interacting protein MDC1, is required for efficient classical NHEJ (C-NHEJ) but with short deletions and insertions. Epistasis analysis revealed that ataxia telangiectasia mutated (ATM) and the chromatin remodeling complex Tip60/TRRAP/P400 are essential for this H2AX function. Taken together, these data suggest that a subset of DSBs may require γH2AX-mediated short-range nucleosome repositioning around the breaks to facilitate C-NHEJ with loss of a few extra nucleotides at NHEJ junctions. This may prevent outcomes such as non-repair and translocations, which are generally more destabilizing to genomes than short deletions and insertions from local NHEJ.

Buried territories: heterochromatic response to DNA double-strand breaks.

Cellular response to DNA double-strand breaks (DSBs), the most deleterious type of DNA damage, is highly influenced by higher-order chromatin structure in eukaryotic cells. Compared with euchromatin, the compacted structure of heterochromatin not only protects heterochromatic DNA from damage, but also adds an extra layer of control over the response to DSBs occurring in heterochromatin. One key step in this response is the decondensation of heterochromatin structure. This decondensation process facilitates the DNA damage signaling and promotes proper heterochromatic DSB repair, thus helping to prevent instability of heterochromatic regions of genomes. This review will focus on the functions of the ataxia telangiectasia mutated (ATM) signaling cascade involving ATM, heterochromatin protein 1 (HP1), Krüppel-associated box (KRAB)-associated protein-1 (KAP-1), tat-interacting protein 60 (Tip60), and many other protein factors in DSB-induced decondensation of heterochromatin and subsequent repair of heterochromatic DSBs. As some subsets of DSBs may be repaired in heterochromatin independently of the ATM signaling, a possible repair model is also proposed for ATM-independent repair of these heterochromatic DSBs.

[Ornithine aspartate and naloxone combined therapy for hepatic encephalopathy affects cognitive function, prognosis, and neuropeptide levels].

OBJECTIVE: To investigate the potential effects on cognitive function, prognosis, and neuropeptide levels of patients in response to combination therapy with ornithine aspartate plus naloxone for hepatic encephalopathy. METHODS: Eighty-four consecutive patients diagnosed with hepatic encephalopathy were randomly divided into two equal groups. The control group (n = 42) received traditional medical treatment, and the research group (n = 42) received the traditional medical treatment as well as the combination therapy with ornithine aspartate plus naloxone. The supplemental treatment was comprised of daily intravenous injection of 10-15 g ornithine aspartate in 250 ml of 5% glucose plus intravenous drip of 3 mg naloxone in 100 ml of 5% glucose, and was given in 7-day cycles for one or two cycles. The cognitive function of patients was assessed by Hasegawa Intelligence Scale (HDS) and Mini-Mental State Examination (MMSE) questionnaires. The effective rate and time duration from coma to consciousness were recorded. Changes in blood ammonia level, markers of liver function, and neuropeptide levels were measured by standard biochemical assays. Intergroup differences were assessed by the Chi-squared test. RESULTS: The HDS and MMSE scores of the research group were significantly higher than those of the control group after therapy. The effective rate, time duration from coma to consciousness, blood ammonia, the liver function markers alanine aminotransferase, gamma-glutamyl-transpeptidase and total bilirubin, and the neuropeptides arginine vasopressin and beta-endorphin were remarkably improved after treatment in the research group, as compared with that in the control group. CONCLUSION: Supplementing the traditional treatment for hepatic encephalopathy with ornithine aspartate plus naloxone combination therapy provides better therapeutic outcome than traditional treatment alone.

The safety and feasibility of laparoscopic right posterior sectionectomy vs. open approach: A systematic review and meta-analysis.

Background: Laparoscopic right posterior sectionectomy (LRPS) is one of the most technically challenging and potentially hazardous procedures in laparoscopic liver resection. Although some available literature works demonstrated the safety and feasibility of LRPS, these data are limited to reports from a single institution and a small sample size without support from evidence-based medicine. So, we performed a meta-analysis to assess further the safety and feasibility of LRPS by comparing it with open right posterior sectionectomy (ORPS). Methods: MEDLINE, Embase, and Cochrane Library were systematically searched for eligible studies comparing LRPS and open approaches. Random and fixed-effects models were used to calculate outcome measures. Results: Four studies involving a total of 541 patients were identified for inclusion: 250 in the LRPS group and 291 in the ORPS group. The postoperative complication and margin were not statistically different between the two groups (OR: 0.49, 95% CI: 0.18 to 1.35, P = 0.17) (MD: 0.05, 95% CI: -0.47 to 0.57, P = 0.86), respectively. LRPS had a significantly longer operative time and shorter hospital stay (MD: 140.32, 95% CI: 16.73 to 263.91, P = 0.03) (MD: -1.64, 95% CI: -2.56 to -0.72, P = 0.0005) respectively. Conclusion: Data from currently available literature suggest that LRPS performed by an experienced surgeon is a safe and feasible procedure in selected patients and is associated with a reduction in the hospital stay.

Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.

BACKGROUND: Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. RESULTS: In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. CONCLUSIONS: Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

The reasonable drainage option after laparoscopic common bile duct exploration for the treatment of choledocholithiasis.

OBJECTIVE: To obtain a reasonable drainage after laparoscopic common bile duct exploration (LCBDE) for the treatment of choledocholithiasis. METHODS: Data of 350 consecutive patients who underwent LCBDE in our hospital from January 2014 to December 2016 were retrospectively reviewed. All the patients were divided into three groups according to different drainage types after LCBDE, including T-tube group with 116 cases, primary closure (PC) group with 114 cases and stent insertion group with 120 cases. Operative parameters and outcomes were compared. RESULTS: The operative time was no significant difference between the T-tube group (106.71 ± 5.19 min), PC group (105.46 ± 5.77 min) and stent insertion group (106.88 ± 5.91 min) (F = 2.175, P = 0.115). The postoperative hospital stay was significantly shorter in the stent insertion group (5.62 ± 0.70 d) than in the T-tube group (7.79 ± 0.85 d) and PC group (7.60 ± 0.80 d) (F = 279.649, P = 0.000). The hospitalization cost was significantly less in the stent insertion group (19,432.78 ± 661.74 yuan) than in the T-tube group (22,059.90 ± 697.98 yuan) and PC group (21,927.20 ± 772.02 yuan) (F = 512.492, P = 0.000). The incidence of postoperative biliary-specific complications was 2.59% (3/116 cases) in the T-tube group, 2.63% (3/114 cases) in the PC group, and 0% (0/120 cases) in the stent insertion group, but this difference was not statistically significant (χ2 = 3.177, P = 0.204). The return to normal levels of postoperative liver function tests (LFTs) was significantly faster in the stent insertion group and T-tube group than in the PC group (P < 0.05). The number of 314 patients were followed up for a median time of 20 months (range from 1-48 months), and no biliary stricture, cholangitis or stone recurrence occurred in these patients during that time. CONCLUSIONS: Stent insertion shows better results when compared with T-tube drainage and primary duct closure in terms of postoperative hospital stay and hospitalization cost. It is the prior option for the choledochotomy closure after LCBDE in suitable patients.

Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing.

BACKGROUND: Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. RESULTS: In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined "3n"-, "3n + 1"-, or "3n + 2"-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to "3n + 1"-bp, "3n + 2"-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. CONCLUSIONS: NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.

Aberrant expression of the autocrine motility factor receptor correlates with poor prognosis and promotes metastasis in gastric carcinoma.

AMFR, autocrine motility factor receptor, also called gp78, is a cell surface cytokine receptor which has a dual role as an E3 ubiquitin ligase in endoplasmic reticulum-associated degradation. AMFR expression is associated with tumor malignancy. We here investigated the clinical significance of AMFR and its role in metastasis and prognosis in gastric cancer. Expression of AMFR, E-cadherin and N-cadherin in cancer tissues and matched adjacent normal tissues from 122 gastric cancer (GC) patients undergoing surgical resection was assessed by immunohistochemistry. Levels of these molecules in 17 cases selected randomly were also analysed by Western blotting. AMFR expression was significantly increased in gastric cancer tissues, and associated with invasion depth and lymph node metastasis. Kaplan-Meier analysis showed AMFR expression correlated with poor overall survival and an increased risk of recurrence in the GC cases. Cox regression analysis suggested AMFR to be an independent predictor for overall and recurrence-free survival. E-cadherin expression was decreased in gastric cancer tissues; conversely, N-cadherin was increased. Expression of AMFR negatively correlated with E-cadherin expression, whereas N-cadherin expression showed a significant positive correlation with AMFR expression. AMFR might be involved in the regulation of epithelial-mesenchymal transition, with aberrant expression correlating with a poor prognosis and promoting invasion and metastasis in GCs.