The present invention provides novel donor polynucleotides formed by linking the two ends of a genomic fragment containing a cleavable site by a polynucleotide carrying a positive selection marker gene and a negative selection marker gene. Use of the donor polynucleotide makes it possible to modify only a target gene with avoiding the possibility of introducing mutations to sequences, called off-target, which are other than the target sequence, by introducing cleavage in a homologous site of the donor polynucleotide without introducing cleavage in a target gene locus.

The present disclosure relates generally to methods of producing rejuvenated T cells, comprising, contacting T cells with at least one reprogramming factor and reactivating the contacted cells; and compositions and methods of using same. The present disclosure also describes cell populations prepared according to methods described herein. The disclosure also provides for methods of treating patients using cell populations prepared by the methods described herein.

The present invention provides donor polynucleotides formed by linking the two ends of a genomic fragment containing a cleavable site by a polynucleotide carrying a positive selection marker gene and a negative selection marker gene. Use of the donor polynucleotide makes it possible to modify only a target gene with avoiding the possibility of introducing mutations to sequences, called off-target, which are other than the target sequence, by introducing cleavage in a homologous site of the donor polynucleotide without introducing cleavage in a target gene locus.