Available Technologies

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Rapid, one-step CRISPR/Cas9-based method for simultaneous bi-allelic editing of a gene

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.





  • Simultaneous bi-allelic gene editing in a single reaction
  • Rapid, one-step creation of cell lines (5-15 days) depending on selection strategy
  • Flexibility in genetic manipulation allowing for essentially any desired alteration of a gene
  • High specificity and accuracy


  • Generation of cell lines for disease modeling, drug screening, clinical applications etc. 
  • Genetic modification of agriculturally-important crops
  • Synthetic biology applications (e.g. engineering microorganisms for biofuel production)
  • Use in basic research on structure and function of protein encoding gene
  • Reengineering patient cells during adoptive cell transfer


Stage of Development:

  • Gene deletions up to 17000bp
  • Gene insertions up to 3600bp
  • Over 20 crated cell lines

Reference Media:

Publication under review


Desired Partnerships:



Docket # 17-8002