Kunyu Li

Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

Regulating the Regulators: microRNA and Asthma

One obstacle to developing an effective therapeutic strategy to treat or prevent asthma is that the fundamental causes of asthma are not totally understood. Asthma is thought to be a chronic TH2 immune-mediated inflammatory disease. Epigenetic changes are recognized to play a role in the initiation and maintenance of a TH2 response. MicroRNAs (miRNAs) are key epigenetic regulators of gene expression, and their expression is highly regulated, therefore, deregulation of miRNAs may play an important role in the pathogenesis of asthma. Profiling circulating miRNA might provide the highest specificity and sensitivity to diagnose asthma; similarly, correcting potential defects in the miRNA regulation network may lead to new therapeutic modalities to treat this disease.

114 A Highly Sensitive and Specific Universal Mirna Profiling Method.

Background miRNAs can be used as robust biomarkers for diagnosis, staging, prognosis and the response to therapy in various diseases. Although a wide spectrum of miRNA detection techniques have been developed, none can accurately and sensitively perform genome-wide high-throughput miRNA profiling (Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, et al 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33:e179). This problem stems from that miRNAs are only ∼22 bases, and multiple species of nucleic acids that contain the mature miRNA sequences are present in the total RNA samples that are usually used for miRNA detection. Methods A novel RT-qPCR miRNA assay (UQmiR, universally quantitating miRNA) was developed to overcome the difficulty. This assay requires only one RT reaction and one universal set of multiple hydrolysis probes to detect all miRNAs, using one universal RT primer, a common reverse primer, and individual miRNA-specific forward primers. A computer program (MSPPD, miRNA-specific primer and probe designer) was developed for the assay. Results The UQmiR has the advantages, but not the disadvantages, of the 2 mostly used miRNA assays. It has the specificity of hydrolysis probe assay and the universal detection of SYBR Green assay. This assay is more sensitive and specific than the commercially available hydrolysis probe assay and SYBR Green assay. Using this method, we have successfully detected 91 out of 96 miRNAs in 0.8 mL of plasma for each miRNA. Conclusions This approach affords a highly specific, sensitive, economical and convenient system to profile the expression of all known miRNAs.