One-step genotyping method in CRISPR based on short inner primer-assisted, tetra primer-paired amplifications.

Base editors and prime editors induce precise DNA modifications over one or several nucleotides in eukaryotic cells. The T7E1 assay has been widely adopted for the assessment of genome editing, but it has several limitations in the applications for prime editing and base editing due to low sensitivity, inaccuracy and additional disadvantages. Here, we propose a short inner primer-assisted, tetra primer-paired amplification (SIPATA) method as an alternative to T7E1 analysis. SIPATA is a PCR-based method in which two long outer and two short (15 nt) inner primers are used for the amplification of a specific genotype in the presence of Hot start-Taq. One of the inner primers carries a 3'-terminally wild-type nucleotide sequence, and the other carries a post-editing sequence. Under optimized conditions, SIPATA enabled sensitive and accurate genotyping of single-nucleotide conversions by base editors and prime editors. Furthermore, SIPATA could be applied to trace low levels of DNA modifications achieved by HDR-mediated gene correction or chimerism during the generation of model animals. Multiplexed genotyping was also possible without compromising those multifaceted analytical advantages of SIPATA. Our findings demonstrate that SIPATA offers a robust, fast and sensitive genotyping platform for single-nucleotide variations in a variety of CRISPR applications.

Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2'-O-methylated uridinylate-rich 3'-overhang in mouse zygotes.

The CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3'-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2'-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.

Aglycosylated antibody-producing mice for aglycosylated antibody-lectin coupled immunoassay for the quantification of tumor markers (ALIQUAT).

Targeting aberrant glycoforms has been validated for in vitro cancer diagnostic development, and several assays are currently in routine clinical use. Because N-glycans in Fc region of antibodies show cross-reactivity with various lectins, high-quality aglycosylated antibodies are exceptionally important for immunoassay platform-based quantitative measurements. Previously, aglycosylated antibody acquisition relied on incomplete, uneconomical and onerous enzymatic and chemical methods. Here, we edited four murine immunoglobulin G genes using adenine base-editing and homology-directed recombination (HDR)-mediated gene editing methods to generate aglycosylated antibody-producing mice. Resulting aglycosylated antibodies showed required analytical performances without compromised protein stability. Thus, this aglycosylated monoclonal antibody-lectin coupled immunoassay for the quantification of tumour markers (ALIQUAT) method can provide a robust, versatile and accessible immunoassay platform to quantify specific glycoforms in precision cancer diagnostics. Moreover, the engineered mice can be used as a host to produce various aglycosylated antibodies in a convenient and robust fashion, thereby expanding in vitro diagnostic development opportunities that utilize glycoforms as a disease-specific biomarkers.

Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3'-overhang.

Genome editing has been harnessed through the development of CRISPR system, and the CRISPR from Prevotella and Francisella 1 (Cpf1) system has emerged as a promising alternative to CRISPR-Cas9 for use in various circumstances. Despite the inherent multiple advantages of Cpf1 over Cas9, the adoption of Cpf1 has been unsatisfactory because of target-dependent insufficient indel efficiencies. Here, we report an engineered CRISPR RNA (crRNA) for highly efficient genome editing by Cpf1, which includes a 20-base target-complementary sequence and a uridinylate-rich 3'-overhang. When the crRNA is transcriptionally produced, crRNA with a 20-base target-complementary sequence plus a U4AU4 3'-overhang is the optimal configuration. U-rich crRNA also maximizes the utility of the AsCpf1 mutants and multiplexing genome editing using mRNA as the source of multiple crRNAs. Furthermore, U-rich crRNA enables a highly safe and specific genome editing using Cpf1 in human cells, contributing to the enhancement of a genome-editing toolbox.

Identification of Mycobacterial Antigens in Human Urine by Use of Immunoglobulin G Isolated from Sera of Patients with Active Pulmonary Tuberculosis.

Point-of-care (POC) diagnostic testing of tuberculosis (TB) is a tremendous unmet need. In this study, four urinary mycobacterial antigens were identified through two independent approaches using IgG capture and immunodepletion methods. Among these, ModC was validated by a multiple reaction monitoring (MRM) method. As expected, the biomarkers elevated the clinical validity of TB diagnosis when combined with preexisting markers.