SUMMARY OF INVENTION
MSK investigators have developed a simple one-step method to build CRISPR/Cas9 libraries expressing paired guide RNAs starting from a pool of short oligonucleotides. This allows for the cloning of specific gRNA-pairs into a single CRISPR expression vector for co-delivery of Cas9 and paired gRNAs, enabling the generation of large-scale pooled gRNA libraries. This method greatly expands the potential applications of CRISPR technology for functional genomics in vitro and in vivo.
The CRISPR-Cas9 genome editing system uses co-expression of the bacterial Cas9 endonuclease and a short guide RNA molecule (gRNA) to disrupt the frame of protein-coding genes and generate loss-of-function alleles. Many applications of the CRISPR-Cas9 system require co-expression of two guide RNAs in a single vector, which is traditionally accomplished by sequential cloning steps. Such a sequential cloning strategy is incompatible with the generation of large-scale pooled gRNA libraries, a challenge that is effectively addressed by MSK’s novel method to build CRISP/Cas9 libraries.
Simple, fast, and inexpensive one-step method to generate large-scale pooled gRNA libraries
Broad applicability as a research tool to generate CRISPR libraries in vitro and in vivo
As an efficient method for generating pooled CRISPR libraries, this novel technology will address a growing demand for use in vivo and in vitro functional genomic studies.
STAGE OF DEVELOPMENT
PCT application PCT/US2016/017377 filed on February 11, 2016
Vidigal, JA, Ventura, A. Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries. Nature Communications (2015). (PMID: 26278926).
Andrea Ventura, MD, PhD, Laboratory Head, Cancer Biology & Genetics Program, Memorial Sloan Kettering
Eileen Flowers, PhD