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Streamlined CRISPR/Cas9 method allowing for differential modification of two alleles of the same gene and generation of new cell lines in a single reaction.
Gene editing using CRISPR/Cas9 system has led to a recent ‘revolution’ in nearly all eukaryotic systems. Most systems where it could be useful (human cells, plants, etc) are diploid, meaning that they have two alleles of every gene. Existing methods for editing both alleles using CRISPR/Cas9 involve targeting one allele at a time which is time-consuming due to the need for screening at each step. These methods are also inefficient at the molecular level because of risk of unwanted side-reactions and errors in the genetic manipulations. There is an immediate need for approaches to streamline the manipulation of both copies of the gene.
Dr. Black’s Lab at Penn has developed an improved CRISPR/Cas9 gene editing method that allows for simultaneous modification of both alleles of a single gene in one transfection, resulting in efficient one-step generation of cell lines with differentially modified alleles of the same gene. With this approach, one could make two very different versions of each allele, e.g. replace one allele and remove/replace another, and make essentially any desired alteration to the gene of interest. Inventors have deleted endogenous genes of 7,000-17,000 base pairs in size and replaced them with genes of 1,100-3,600 base pair size range. Inventors believe that these ranges can be further extended.
Ben E. Black, Ph.D.
Stage of Development:
Publication under review
Docket # 17-8002