Guide-specific loss of efficiency and off-target reduction with Cas9 variants.

High-fidelity clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) variants have been developed to reduce the off-target effects of CRISPR systems at a cost of efficiency loss. To systematically evaluate the efficiency and off-target tolerance of Cas9 variants in complex with different single guide RNAs (sgRNAs), we applied high-throughput viability screens and a synthetic paired sgRNA-target system to assess thousands of sgRNAs in combination with two high-fidelity Cas9 variants HiFi and LZ3. Comparing these variants against wild-type SpCas9, we found that ∼20% of sgRNAs are associated with a significant loss of efficiency when complexed with either HiFi or LZ3. The loss of efficiency is dependent on the sequence context in the seed region of sgRNAs, as well as at positions 15-18 in the non-seed region that interacts with the REC3 domain of Cas9, suggesting that the variant-specific mutations in the REC3 domain account for the loss of efficiency. We also observed various degrees of sequence-dependent off-target reduction when different sgRNAs are used in combination with the variants. Given these observations, we developed GuideVar, a transfer learning-based computational framework for the prediction of on-target efficiency and off-target effects with high-fidelity variants. GuideVar facilitates the prioritization of sgRNAs in the applications with HiFi and LZ3, as demonstrated by the improvement of signal-to-noise ratios in high-throughput viability screens using these high-fidelity variants.

Guide-specific loss of efficiency and off-target reduction with Cas9 variants.

High-fidelity Cas9 variants have been developed to reduce the off-target effects of CRISPR systems at a cost of efficiency loss. To systematically evaluate the efficiency and off-target tolerance of Cas9 variants in complex with different single guide RNAs (sgRNAs), we applied high-throughput viability screens and a synthetic paired sgRNA-target system to assess thousands of sgRNAs in combination with two high-fidelity Cas9 variants HiFi and LZ3. Comparing these variants against WT SpCas9, we found that ~20% of sgRNAs are associated with a significant loss of efficiency when complexed with either HiFi or LZ3. The loss of efficiency is dependent on the sequence context in the seed region of sgRNAs, as well as at positions 15-18 in the non-seed region that interacts with the REC3 domain of Cas9, suggesting that the variant-specific mutations in REC3 domain account for the loss of efficiency. We also observed various degrees of sequencedependent off-target reduction when different sgRNAs are used in combination with the variants. Given these observations, we developed GuideVar, a transfer-learning-based computational framework for the prediction of on-target efficiency and off-target effect with high-fidelity variants. GuideVar facilitates the prioritization of sgRNAs in the applications with HiFi and LZ3, as demonstrated by the improvement of signal-to-noise ratios in high-throughput viability screens using these high-fidelity variants.

[Analysis of Heavy Metal Sources in Groundwater and Assessment of Health Risks: An Example from the Southwest Sub-basin of the Shiqi River].

In order to explore the sources of pollution and health risk profile of heavy metal elements in groundwater, 41 sets of representative groundwater samples from the southwest sub-basin of the Shiqi River were examined for 10 heavy metal elements (As, Cr, Cd, Al, Cu, Zn, Ni, Co, Mn, and Hg), and correlation analysis and principal component analysis were used to resolve the possible sources of heavy metal contamination in groundwater in the study area. The concentration characteristics and health risk levels of the 10 heavy metals were assessed using the single-factor contamination index (Pi), the Nemerow comprehensive contamination index (PN), and the health risk model. The results showed that:① the average values of heavy metal elements of the groundwater in the study area all met the limit of the class Ⅲ water standard in the quality standard for groundwater (GB/T 14848-2017); only the maximum value of Al was exceeded, followed by a large variation in the concentrations of Al, Mn, and Cr. The heavy metal element with the largest average contribution was Al (65.74%). ② The results of the single-factor contamination index evaluation showed that only the heavy metal element Al exceeded the cleaning level, and the results of the Nemerow comprehensive contamination index evaluation showed that the study area was basically at low pollution levels, and the quality of groundwater was good. ③ The results of the multivariate statistical analysis showed that Zn, Co, and Mn were from mixed sources consisting of geological formation and domestic waste; Al, As, and Cu were from agricultural sources; Cd, Cr, and Ni were from industrial sources; and Hg came from long-range atmospheric transport. ④ The health risk values for all heavy metals in the study area were within acceptable limits, with higher health risk values for children than for adults from the drinking water route, lower health risk values than in adults from the dermal route, and higher health risk values for heavy metals from the drinking water route than those from the dermal route, indicating that the drinking water route was the main route of exposure to heavy metals.

Spatial Distribution, Source Analysis and Health Risk Study of Heavy Metals in the Liujiang River Basin in Different Seasons.

Three high-frequency sampling and monitoring experiments were performed at the Lutang and Luowei transects of the Liujiang River entrance and at the southeast exit of the Liuzhou during 2019 for the purpose of assessing physico-chemical variables and human health hazards of water heavy metals in different rainfall processes. There were significant seasonal variations in concentrations of 11 heavy metals and most variables showed higher levels during the dry season. The distribution of heavy metals in the Liuzhou area varied significantly by region. Pollution source analysis indicated distinct seasons of wetness and dryness. The dry season is dominated by anthropogenic activities, while the wet season is dominated by natural processes. The results of hazard quotient (HQ) and carcinogenic risk (CR) analysis showed that the health risk of non-carcinogenic heavy metals in the wet season is slightly higher than that in the dry season. Seasonal changes in carcinogenic risk are the opposite; this is due to the combined influence of natural and human activities on the concentration of heavy metals in the river. Among them, Al was the most important pollutant causing non-carcinogenic, with As being a significant contributor to carcinogenic health risk. Spatially, the downstream Luowei transect has a high health risk in both the dry and rainy seasons, probably due to the fact that the Luowei transect is located within a major industrial area in the study area. There are some input points for industrial effluent discharge in the area. Therefore, high-frequency monitoring is essential to analyze and reduce the heavy metal concentrations in the Liujiang River during dry and wet seasons in order to protect the health of the residents in the area.

Health Risk Assessment Based on Source Identification of Heavy Metal(loid)s: A Case Study of Surface Water in the Lijiang River, China.

In this study, 24 surface water samples were collected from the main trunk/tributary of the Lijiang River during the wet season (April) and the dry season (December) in 2021. The total concentration of 11 heavy metal(loid)s (Al, Cu, Pb, Zn, Cr, Ni, Co, Cd, Mn, As, and Hg) was determined to investigate their physicochemical properties and spatial-temporal distribution characteristics. The heavy metal evaluation index (HEI) and the positive matrix factorization (PMF) model were employed to evaluate water quality and to reveal quantitatively identified pollution sources for further investigation to obtain a health risk assessment using the hazard index (HI) and carcinogenic risk (CR) of various pollution sources. The mean concentrations of heavy metal(loid)s in surface water in the wet and dry seasons were ranked as: Al > Mn > Zn > Ni > Cd > Cr > Cu > As >Hg = Pb > Co, with the mean concentration of Hg being higher than the national Class II surface water environmental quality standard (GB3838-2002). In terms of time scale, the concentration of most heavy metal(loid)s was higher in the wet season; most heavy metal(loid)s were distributed mainly in the midstream area. HEI index indicated that the main water quality status was "slightly affected" in the study area. Five potential sources of pollution were obtained from the PMF model, including industrial activities, traffic sources, agricultural activities, domestic waste emissions, and natural resources. The source-oriented risk assessment indicated that the largest contributions of HI and CR were agricultural sources in the Lijiang River. This study provides a "target" for the precise control of pollution sources, which has a broad impact on improving the fine management of the water environment in the basin.

Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity.

The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. To systematically explore the sequence determinants governing CRISPR/Cas9 specificity, here we describe a dual-target system to measure the relative cleavage rate between off- and on-target sequences (off-on ratios) of 1902 gRNAs on 13,314 synthetic target sequences, and reveal a set of sequence rules involving 2 factors in off-targeting: 1) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context; 2) an "epistasis-like" combinatorial effect of multiple mismatches, which are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. These sequence rules lead to the development of MOFF, a model-based predictor of Cas9-mediated off-target effects. Moreover, the "epistasis-like" combinatorial effect suggests a strategy of allele-specific genome editing using mismatched guides. With the aid of MOFF prediction, this strategy significantly improves the selectivity and expands the application domain of Cas9-based allele-specific editing, as tested in a high-throughput allele-editing screen on 18 cancer hotspot mutations.

The RNA helicase Dhx15 mediates Wnt-induced antimicrobial protein expression in Paneth cells.

RNA helicases play roles in various essential biological processes such as RNA splicing and editing. Recent in vitro studies show that RNA helicases are involved in immune responses toward viruses, serving as viral RNA sensors or immune signaling adaptors. However, there is still a lack of in vivo data to support the tissue- or cell-specific function of RNA helicases owing to the lethality of mice with complete knockout of RNA helicases; further, there is a lack of evidence about the antibacterial role of helicases. Here, we investigated the in vivo role of Dhx15 in intestinal antibacterial responses by generating mice that were intestinal epithelial cell (IEC)-specific deficient for Dhx15 (Dhx15 f/f Villin1-cre, Dhx15ΔIEC). These mice are susceptible to infection with enteric bacteria Citrobacter rodentium (C. rod), owing to impaired α-defensin production by Paneth cells. Moreover, mice with Paneth cell-specific depletion of Dhx15 (Dhx15 f/f Defensinα6-cre, Dhx15ΔPaneth) are more susceptible to DSS (dextran sodium sulfate)-induced colitis, which phenocopy Dhx15ΔIEC mice, due to the dysbiosis of the intestinal microbiota. In humans, reduced protein levels of Dhx15 are found in ulcerative colitis (UC) patients. Taken together, our findings identify a key regulator of Wnt-induced α-defensins in Paneth cells and offer insights into its role in the antimicrobial response as well as intestinal inflammation.

Author Correction: De novo identification of essential protein domains from CRISPR-Cas9 tiling-sgRNA knockout screens.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

De novo identification of essential protein domains from CRISPR-Cas9 tiling-sgRNA knockout screens.

High-throughput CRISPR-Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. Here, to facilitate de novo identification of essential protein domains from such screens, we propose ProTiler, a computational method for the robust mapping of CRISPR knockout hyper-sensitive (CKHS) regions, which refer to the protein regions associated with a strong sgRNA dropout effect in the screens. Applied to a published CRISPR tiling screen dataset, ProTiler identifies 175 CKHS regions in 83 proteins. Of these CKHS regions, more than 80% overlap with annotated Pfam domains, including all of the 15 known drug targets in the dataset. ProTiler also reveals unannotated essential domains, including the N-terminus of the SWI/SNF subunit SMARCB1, which is validated experimentally. Surprisingly, the CKHS regions are negatively correlated with phosphorylation and acetylation sites, suggesting that protein domains and post-translational modification sites have distinct sensitivities to CRISPR-Cas9 mediated amino acids loss.

[Simultaneous determination of chlorogenic acid and cynaroside contents in Lonicerae Japonica Flos by high resolution sampling two-dimensional liquid chromatography].

An innovative analytical method based on high resolution sampling two-dimensional liquid chromatography (HiRes 2D-LC) was established for determination of chlorogenic acid and cynaroside in Lonicerae Japonica Flos. A C18 column was used in the first dimension (1D)-LC separation with acetonitrile and 0.4% (v/v) phosphoric acid aqueous solution as mobile phases. Five heart cuts of chlorogenic acid and four heart cuts of cynaroside were stored in 2D-LC interface, which was a 5-position-10-port valve equipped with two multiple heart-cutting valves. The stored cuts were sequentially separated in the second dimension (2D)-LC. The 2D separation was carried out on an SB-Phenyl column with acetonitrile and 0.5% (v/v) acetic acid aqueous solution as mobile phases. The results showed that chlorogenic acid peaks in the 1D were well separated, whereas cynaroside peaks in the 1D were co-eluted with interferences. The above two targets were accurately quantified through a high resolution sampling mode based on continuous slice cuts of the whole target peaks. The method had good linearity, recovery and repeatability. The HiRes 2D-LC system could be used to improve separation and quantification of (un)targets in traditional Chinese medicine samples.

DNMT1-maintained hypermethylation of Krüppel-like factor 5 involves in the progression of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the major subtype of renal cell carcinoma (RCC) that is resistant to conventional radiation and chemotherapy. It is a challenge to explore effective therapeutic targets and drugs for this kind of cancer. Transcription factor Krüppel-like factor 5 (KLF5) exerts diverse functions in various tumor types. By analyzing cohorts of the Cancer Genome Atlas (TCGA) data sets, we find that KLF5 expression is suppressed in ccRCC patients and higher level of KLF5 expression is associated with better prognostic outcome. Our further investigations demonstrate that KLF5 genomic loci are hypermethylated at proximal exon 4 and suppression of DNA methyltransferase 1 (DNMT1) expression by ShRNAs or a methylation inhibitor 5-Aza-CdR can recover KLF5 expression. Meanwhile, there is a negative correlation between expressions of KLF5 and DNMT1 in ccRCC tissues. Ectopic KLF5 expression inhibits ccRCC cell proliferation and migration/invasion in vitro and decreases xenograft growth and metastasis in vivo. Moreover, 5-Aza-CdR, a chemotherapy drug as DNMTs' inhibitor that can induce KLF5 expression, suppresses ccRCC cell growth, while knockdown of KLF5 abolishes 5-Aza-CdR-induced growth inhibition. Collectively, our data demonstrate that KLF5 inhibits ccRCC growth as a tumor suppressor and highlight the potential of 5-Aza-CdR to release KLF5 expression as a therapeutic modality for the treatment of ccRCC.

Inhibition of Snail Family Transcriptional Repressor 2 (SNAI2) Enhances Multidrug Resistance of Hepatocellular Carcinoma Cells.

China accounts for almost half of the total number of liver cancer cases and deaths worldwide, and hepatocellular carcinoma (HCC) is the most primary liver cancer. Snail family transcriptional repressor 2 (SNAI2) is known as an epithelial to mesenchymal transition-inducing transcription factor that drives neoplastic epithelial cells into mesenchymal phenotype. However, the roles of endogenous SNAI2 remain controversial in different types of malignant tumors. Herein, we surprisingly identify that anchorage-independent growth, including the formation of tumor sphere and soft agar colony, is significantly increased when SNAI2 expression is inhibited by shRNAs in HCC cells. Suppression of SNAI2 suffices to up-regulate several cancer stem genes. Although unrelated to the metastatic ability, SNAI2 inhibition does increase the efflux of Hoechst 33342 and enhance multidrug resistance in vitro and in vivo. In agreement with this data, we demonstrate for the first time that decreasing SNAI2 level can transcriptionally upregulate several ATP binding cassette (ABC) transporter genes such as ABCB1. Moreover, ABC transporters' inhibitor verapamil can rescue the multidrug resistance induced by SNAI2 inhibition. Our results implicate that SNAI2 behaves as a tumor suppressor by inhibiting multidrug resistance via suppressing ABC transporter genes in HCC cells.