CRISPR Based Genomic Editing

CRISPR technology enables researchers to use customizable RNA guides to genetically modify the genome, control gene expression, or perform live cell imaging of specific genomic loci. CRISPR technology originates from an immune defense mechanism in prokaryotes. Engineering of the bacteria CRISPR system has led to much simplified molecular tools that can be readily applied in mammalian cells.

Currently, the most commonly used CRISPR system is based on that from Streptococcus pyogenes, composing primarily of two components: a protein named Cas9 (or its variants) as well as a small RNA named single guide RNA (sgRNA). Both components can be readily delivered to mammalian hematopoietic cells through either viral infection or plasmid transfection. In addition, for genomic engineering through homologous recombination, a third component of template DNA is needed, which can be delivered through transfection. Besides direct delivery into hematopoietic cells, CRISPR technology has been applied to quickly generate mouse models by delivering CRISPR components into single-cell embryos.

The following are possible with CRISPR technology:

1. Genetic knockout through non-homologous end joining based random mutagenesis.
2. Defined genomic alterations through homologous recombination
3. Control gene activation or repression
4. Live cell imaging of specific genomic loci

YCCEH provides service to advice experimental planning using CRISPR and provides CRISPR-related protocols upon request.

Please contact Core A, Jun Lu, at jun.lu@yale.edu.