Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach

Raj Chari, Prashant Mali, Mark Moosburner, George M Church
Source: Nat Methods
Publication Date: (2015)
Issue: 12(9): 823-6
Cells used in publication:
Species: human
Tissue Origin: blood
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit
For K562 and PGP1 induced pluripotent stem (iPS) cells, DNA was transfected using the Lonza 4D Nucleofector.
We developed an in vivo library-on-library methodology to simultaneously assess single guide RNA (sgRNA) activity across ~1,400 genomic loci. Assaying across multiple human cell types and end-processing enzymes as well as two Cas9 orthologs, we unraveled underlying nucleotide sequence and epigenetic parameters. Our results and software (http://crispr.med.harvard.edu/sgRNAScorer) enable improved design of reagents, shed light on mechanisms of genome targeting, and provide a generalizable framework to study nucleic acid-nucleic acid interactions and biochemistry in high throughput.