Adapting CRISPR/Cas9 for functional genomics screens.
The use of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) for targeted genome editing has been widely adopted and is considered a "game changing" technology. The ease and rapidity by which this approach can be used to modify endogenous loci in a wide spectrum of cell types and organisms makes it a powerful tool for customizable genetic modifications as well as for large-scale functional genomics. The development of retrovirus-based expression platforms to simultaneously deliver the Cas9 nuclease and single guide (sg) RNAs provides unique opportunities by which to ensure stable and reproducible expression of the editing tools and a broad cell targeting spectrum, while remaining compatible with in vivo genetic screens. Here, we describe methods and highlight considerations for designing and generating sgRNA libraries in all-in-one retroviral vectors for such applications.
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Abba
A
Malina
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Alexandra
A
Katigbak
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Regina
R
Cencic
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Rayelle Itoua
RI
Maïga
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Francis
F
Robert
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Hisashi
H
Miura
Department of Biochemistry, McGill University, Montreal, Quebec, Canada; Department of Oncology, McGill University, Montreal, Quebec, Canada; The Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, Quebec, Canada. Electronic address: jerry.pelletier@mcgill.ca.
Jerry
J
Pelletier
D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005820:Genetic Testing / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000008:administration & dosage / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems
CRISPR; Cas9; Functional genomics; Genetic screening; Retroviral vectors