Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries.
The CRISPR-Cas9 system is a powerful tool to edit eukaryotic genomes that has recently been adapted for functional screens. Several of its applications--including the disruption of genes using Cas9-nickase and the generation of large deletions--require co-expression of two distinct guide RNAs (gRNAs). However, the lack of experimental approaches to generate pools of paired gRNA vectors prevents these applications from being scalable. Here we report a simple, inexpensive, one-step method that allows for the rapid and efficient cloning of gRNA pairs into expression vectors. We show that this method can be used to generate pooled libraries and is therefore suitable for in vivo and in vitro functional screens.
Memorial Sloan Kettering Cancer Center, Cancer Biology and Genetics Program, 1275 York Avenue, New York, New York 10065, USA.
Joana A
0000000327656985
JA
Vidigal
Memorial Sloan Kettering Cancer Center, Cancer Biology and Genetics Program, 1275 York Avenue, New York, New York 10065, USA.
Andrea
A
Ventura
D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D016159:Tumor Suppressor Protein p53