Abstract: A donor construct and a gene knockout method, as well as a system and kit for the gene knockout are provided. The donor construct is a linear donor DNA or can be cleaved in a cell to produce the linear donor DNA. The gene knockout method uses a marker gene contained in the donor construct to enrich cells in which a gene is knocked out, thereby improving the efficiency of generating the gene knockout by a sequence-specific nuclease.

Abstract: A novel preparation method for a universal CAR-T cell targeting a T-cell lymphoma cell, the universal CAR-T cell prepared by means of the method and a biological product comprising the universal CAR-T cell. The preparation method for the universal CAR-T cell targeting a T-cell lymphoma cell comprises: obtaining a T cell from a human donor having a healthy lymphatic system, and then disrupting a TRAC genome region and a B2M genome region in the T cell by means of a gene editing technology, so that CAR molecules targeting the T-cell lymphoma cell TCR?/? are stably expressed in the T cell. The universal CAR-T cell targeting a T-cell lymphoma cell prepared by means of the preparation method eliminates the natural TCR expression of a T cell, greatly reduces the graft-versus-host reaction, and meanwhile, also greatly reduces the immunogenicity thereof, and can continuously and efficiently kill the T-cell lymphoma cell.

Abstract: A method for purifying a universal human CAR-T cell, comprising: (i) destroying, by using gene editing technology, a TRAC gene region from position 23016448 to position 23016490 of chromosome 14 and a B2M gene region from position 45003745 to position 4500378 of chromosome 15 in the human CAR-T cell; (ii) then introducing a mRNA targeting TCR?/? into the gene-edited CAR-T cell; and (iii) in vitro culturing the CAR-T cell population after the described treatment.

Abstract: Compositions, kits and methods are provided for genetic screening using one or more sets of guide RNA constructs having internal barcodes (“iBAR”). Each set has three or more guide RNA constructs targeting the same genomic locus, but embedded with different iBAR sequences.