Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line

The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein’s catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5–2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.

Re-Sequencing and Transcriptomic Analysis Reveal Differences in Nitrite Reductase in Jujube Fruit (Ziziphus Jujube)

BackgroundJujube is one of the characteristic fruit tree species in China. ‘Linhuang No. 1’, a cracking-resistant cultivar, and ‘Muzao’, a cracking-susceptible cultivar, were selected as materials by previous study. Whole-genome re-sequencing and transcriptome of ‘Linhuang No. 1’ and ‘Muzao’ allow the screening out of differently expressed genes with different gene structures between them. It could be helpful in explaining divergence/similarity between the two cultivars. ResultsThere are 664,129 mutation sites between ‘Linhuang No. 1’ and ‘Muzao’ by re-sequencing. To determine the genetic relationship of ‘Linhuang 1’, ‘Muzao’ and reference genome ‘Dongzao’, the characteristic mutation sites were analyzed by principal component analysis. The genetic relationship between ‘Linhuang No. 1’ and ‘Muzao’ was closer than that with ‘Dongzao’. 19 differentially expressed genes were screened by combining the transcriptomics with re-sequencing analysis. LOC107427052 (encoding nitrite reductase) was determined by KEGG enrichment analysis for further study. The large base insertion was not in the domain region of the LOC107427052 gene CDS region. As verified by the finding that the base insertion did not affect protein translation. LOC107427052 gene expression levels, the nitrite reductase activities and the nitrite content of ‘Muzao’ were significantly higher than those of ‘Linhuang No. 1’ at young fruit stage. There was no significant difference in the product ammonia of nitrite reductase between the two varieties. ConclusionsOur study has laid a foundation for the analysis of genetic information and the comparative nitrite metabolism of ‘Linhuang No. 1’ and ‘Muzao’.

Re-sequencing and transcriptomic analysis reveals rich DNA sequence variation and differential gene expression between varieties of Ziziphus jujuba

Background: Jujube is one of the characteristic fruit tree species in China. ‘Linhuang No. 1’, a cracking-resistant cultivar, and ‘Muzao’, a cracking-susceptible cultivar, were selected as materials by previous study. Whole-genome re-sequencing and transcriptome of ‘Linhuang No. 1’ and ‘Muzao’ allow the screening out of differently expressed genes with different gene structures between them. It could be helpful in explaining divergence/similarity of cracking resistance between the two cultivars. Results: There are 664,129 mutation sites between ‘Linhuang No. 1’ and ‘Muzao’ by re-sequencing. To determine the genetic relationship of ‘Linhuang 1’, ‘Muzao’ and reference genome ‘Dongzao’, the characteristic mutation sites were analyzed by principal component analysis. The genetic relationship between ‘Linhuang No. 1’ and ‘Muzao’ was closer than that with ‘Dongzao’. A total of 431 differentially expressed genes was screened by transcriptomics, and 19 differentially expressed genes were screened by combining the transcriptomics with re-sequencing analysis. LOC107427052 (encoding nitrite reductase) was determined by KEGG enrichment analysis for further study. Conclusions: The large base insertion was not in the domain region of the LOC107427052 gene CDS region. As verified by the finding that the base insertion did not affect protein translation. Our study has laid a foundation for the analysis of genetic information and the comparative nitrite metabolism of ‘Linhuang No. 1’ and ‘Muzao’.

Efficiency of Recombinant CRISPR/rCas9-Mediated miRNA Gene Editing in Rice

Drought is one of the major environmental stresses adversely affecting crop productivity worldwide. Precise characterization of genes involved in drought response is necessary to develop new crop varieties with enhanced drought tolerance. Previously, we identified 66 drought-induced miRNAs in rice plants. For the further functional investigation of the miRNAs, we applied recombinant codon-optimized Cas9 (rCas9) for rice with single-guide RNAs specifically targeting mature miRNA sequences or sites required for the biogenesis of mature miRNA. A total of 458 T0 transgenic plants were analyzed to determine the frequency and type of mutations induced by CRISPR/rCas9 on 13 independent target miRNAs. The average mutation frequency for 13 genes targeted by single guide RNAs (sgRNAs) in T0 generation was 59.4%, including mono-allelic (8.54%), bi-allelic (11.1%), and hetero-allelic combination (39.7%) mutations. The mutation frequency showed a positive correlation with Tm temperature of sgRNAs. For base insertion, one base insertion (99%) was predominantly detected in transgenic plants. Similarly, one base deletion accounted for the highest percentage, but there was also a significant percentage of cases in which more than one base was deleted. The deletion of more than two bases in OsmiR171f and OsmiR818b significantly reduced the level of corresponding mature miRNAs. Further functional analysis using CRISPR/Cas9-mediated mutagenesis confirmed that OsmiR818b is involved in drought response in rice plants. Overall, this study suggests that the CRISPR/rCas9 system is a powerful tool for loss-of-function analysis of miRNA in rice.

Novel quinolone resistance determinant, qepA8, in Shigella flexneri isolated in the United States, 2016

A qepA8+ Shigella flexneri was cultured from the stool of a traveler returning from India and East Asia. This chromosomally encoded qepA variant, has a six-base insertion, and may have been mobilized as part of a complex IS1-mediated composite transposon including catA1, aadA1, and blaOXA-1. In laboratory E. coli, qepA8 alone only conferred decreased ciprofloxacin susceptibility; however, it may work in combination with additional mechanisms to confer clinical resistance.