OliTag-seq enhances in cellulo detection of CRISPR-Cas9 off-targets.

The potential for off-target mutations is a critical concern for the therapeutic application of CRISPR-Cas9 gene editing. Current detection methodologies, such as GUIDE-seq, exhibit limitations in oligonucleotide integration efficiency and sensitivity, which could hinder their utility in clinical settings. To address these issues, we introduce OliTag-seq, an in-cellulo assay specifically engineered to enhance the detection of off-target events. OliTag-seq employs a stable oligonucleotide for precise break tagging and an innovative triple-priming amplification strategy, significantly improving the scope and accuracy of off-target site identification. This method surpasses traditional assays by providing comprehensive coverage across various sgRNAs and genomic targets. Our research particularly highlights the superior sensitivity of induced pluripotent stem cells (iPSCs) in detecting off-target mutations, advocating for using patient-derived iPSCs for refined off-target analysis in therapeutic gene editing. Furthermore, we provide evidence that prolonged Cas9 expression and transient HDAC inhibitor treatments enhance the assay's ability to uncover off-target events. OliTag-seq merges the high sensitivity typical of in vitro assays with the practical application of cellular contexts. This approach significantly improves the safety and efficacy profiles of CRISPR-Cas9 interventions in research and clinical environments, positioning it as an essential tool for the precise assessment and refinement of genome editing applications.

Improved and Flexible HDR Editing by Targeting Introns in iPSCs.

Highly efficient gene knockout (KO) editing of CRISPR-Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.

Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion.

BACKGROUND: After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences. RESULTS: We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%. CONCLUSIONS: Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.

HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs.

Genome-edited human induced pluripotent stem cells (iPSCs) hold great promise for therapeutic applications. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair (HDR)-edited iPSC lines, particularly for silent genes. This is partially due to chromatin compaction, inevitably limiting Cas9 access to the target DNA. Among the six HDAC inhibitors we examined, vorinostat, or suberoylanilide hydroxamic acid (SAHA), led to the highest HDR efficiency at both open and closed loci, with acceptable toxicity. HDAC inhibitors equally increased non-homologous end joining (NHEJ) editing efficiencies (∼50%) at both open and closed loci, due to the considerable HDAC inhibitor-mediated increase in Cas9 and sgRNA expression. However, we observed more substantial HDR efficiency improvement at closed loci relative to open chromatin (2.8 vs. 1.7-fold change). These studies provide a new strategy for HDR-editing of silent genes in iPSCs.

Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.

Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse.

BACKGROUND: Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). RESULTS: We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. CONCLUSIONS: These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.

Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression.

Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of BCL-XL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency. Treatment with a BCL inhibitor ABT-263 further improves HDR efficiency by 70% and KO efficiency by 40%. The increased genome editing efficiency is attributed to higher expressions of Cas9 and sgRNA in surviving cells after electroporation. HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene. Moreover, KO efficiency of 100% can be achieved in a bulk population of cells with biallelic HDR KO followed by double selection, abrogating the necessity for single cell cloning. Taken together, these simple yet highly efficient editing strategies provide useful tools for applications ranging from manipulating human iPSC genomes to creating gene-modified animal models.

Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.

BACKGROUND: Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs). RESULTS: Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97-100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs. CONCLUSIONS: Taken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.

[Influence of chronic lead exposure in rats during the developmental stage on expression of leptin in plasma, cerebrospinal fluid, and hippocampus].

OBJECTIVE: To investigate the influence of lead exposure in rats during the developmental stage on the expression of leptin in plasma, cerebrospinal fluid, and hippocampus, as well as investigating whether leptin is associated with the mechanism of cognitive impairment induced by lead exposure. METHODS: The rat model of cognitive impairment after chronic lead exposure was established by adding lead acetate into drinking water. According to the concentration of lead acetate in drinking water, the rats were divided into control (0 ppm), low-lead (50 ppm), medium-lead (200 ppm), and high-lead groups (1 000 ppm), with 16 rats in each group. Atomic absorption spectrometry was used to measure the content of lead in the plasma, cerebrospinal fluid and hippocampus. ELISA was used to measure the level of leptin in the plasma and cerebrospinal fluid. Immunohistochemistry was used to observe the distribution of leptin protein in the hippocampus. Western blot was used for relative quantification of leptin proteins in the hippocampus. RESULTS: Compared with the control group, the lead exposure groups showed significant increases in the content of lead in blood, cerebrospinal fluid, and hippocampus (P<0.01), as well as significant reductions in the levels of leptin in plasma and cerebrospinal fluid (P<0.05). The results of immunohistochemical staining showed that leptin was mainly distributed in the cytoplasm of pyramidal neurons in the hippocampal CA region. The results of Western blot showed that compared with the control group, the three lead exposure groups showed a slight increase in the protein expression of leptin in the hippocampus (P>0.05). CONCLUSIONS: Lead exposure can reduce the levels of leptin in plasma and cerebrospinal fluid in rats, which may be associated with the mechanism of cognitive impairment induced by lead exposure.

Validity and reliability of the Taiwanese version of the Pittsburgh Sleep Quality Index in cancer patients.

BACKGROUND: Sleep disturbance remains a common symptom among cancer patients. Assessment of sleep disturbance in cancer patients is hindered by infrequent use of standardized tools for sleep management. OBJECTIVES: The purposes of this study were to validate the Taiwanese version of the Pittsburgh Sleep Quality Index (PSQI-T) and to determine detection cut-off points in cancer patients. DESIGN: A cross-sectional and descriptive correlational design. PARTICIPANTS: A sample of 205 Taiwanese patients with various cancer diagnoses. METHODS: The survey included the Pittsburgh Sleep Quality Index-Taiwanese version, the MD Anderson Symptom Inventory-Taiwanese version, the Brief Fatigue Inventory-Taiwanese version, the seven-day sleep log, and Manual of Mental Disorders, Fourth Edition. RESULTS: The internal consistency Cronbach's alpha for the PSQI was 0.79. Test-retest reliability was 0.91 for the global score over a 20- to 28-day interval in a sample of 16 patients. Construct validity was established by a significant relationship of the PSQI-T global score to the total symptom severity score and the fatigue severity score. Convergent validity was examined by correlating the PSQI-T scores and scores of the DSM-IV and scores on the seven-day sleep log. Known-group validity was established by comparing PSQI-T scores for patients having low fatigue levels and those having high fatigue levels. Receiver-operating characteristic (ROC) curves were used to determine the detection cut-off points. CONCLUSIONS: We found that a PSQI-T global score of 8 generates the best sensitivity and specificity for measuring sleep disturbance in cancer patients. The PSQI-T is a reliable, valid, and sensitive instrument for measuring sleep quality among Taiwanese cancer patients.

[Regulation effects of grafting on cinnamic acid and vanillin in eggplant root exudates].

Cinnamic acid and vanillin are the allelochemicals commonly existed in eggplant root exudates. With pot culture experiment, the regulation effects of grafting on the cinnamic acid and vanillin in eggplant root exudates were studied, and the results showed that grafting decreased the amount of the two substances, especially of vanillin, in eggplants root system. The maximum reduction amount of cinnamic acid reached 68.96%, and that of vanillin reached 100%. Under the stress of exotic cinnamic acid and vanillin, especially of exotic cinnamic acid, grafting relieved the autotoxicity of the two substances on eggplants. Compared with own-rooted eggplant, grafted eggplant had a higher plant height and a larger stem diameter, its leaf chlorophyll content increased by 5.26%-13.12%, root electric conductivity and MDA content decreased, and root SOD activity enhanced.