[Donor DNA Modification with Cas9 Targeting Sites Improves the Efficiency of MTC34 Knock-in into the CXCR4 Locus].

To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA.

[Methods to Increase the Efficiency of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the CXCR4 Locus in the CEM/R5 T Cell Line].

The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus.

Efficient Genome Editing Using 'NanoMEDIC' AsCas12a-VLPs Produced with Pol II-Transcribed crRNA.

Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble in the cytoplasm, but U6-driven sgRNA is localized in the nucleus, which hinders the efficient formation and packaging of RNPs into VLPs.

A New Chimeric Antibody against the HIV-1 Fusion Inhibitory Peptide MT-C34 with a High Affinity and Fc-Mediated Cellular Cytotoxicity.

Peptides from heptad repeat (HR1 and HR2) regions of gp41 are effective inhibitors of HIV-1 entry that block the fusion of viral and cellular membranes, but the generation of antibodies highly specific for these peptides is challenging. We have previously described a mouse hybridoma that recognizes MT-C34-related peptides derived from HR2. It was used for the selection of HIV-1-resistant CD4 lymphocytes engineered to express the MT-C34 peptide via a CRISPR/Cas9-mediated knock-in into the locus.

Lymphocyte Phosphatase-Associated Phosphoprotein (LPAP) as a CD45 Protein Stability Regulator.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a binding partner of the phosphatase CD45, but its function remains poorly understood. Its close interaction with CD45 suggests that LPAP may potentially regulate CD45, but direct biochemical evidence for this has not yet been obtained. We found that in the Jurkat lymphoid cells the levels of LPAP and CD45 proteins are interrelated and well correlated with each other.