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

Nat Methods. 2015 Sep;12(9):823-6. doi: 10.1038/nmeth.3473. Epub 2015 Jul 13.


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.

MeSH terms

  • Chromosome Mapping / methods*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Genetic Engineering / methods*
  • Genome, Human / genetics*
  • Genomic Library*
  • Humans
  • RNA / genetics*
  • Software


  • RNA