A non-rational approach to optimized targeting of CRISPR-Cas9 complexes

Abstract

In this issue of Cell Chemical Biology, Bush et al.¹ report an in vitro selection method for optimizing CRISPR-Cas9 single-guide RNAs. This approach may be useful in targeting previously intractable genomic sequences. The results also provide insights into which positions in single-guide RNAs are most amenable to modification.

Figure 1.. sgRNAs are prone to unfavorable intramolecular interactions that inhibit proper loading into Cas9

(A) Proper folding of crRNA domains (blue), tracrRNA domains (green), and engineered tetraloop (red) of the sgRNA allows Cas9-sgRNA ribonucleoprotein (RNP) complex formation with a PAM (purple) and DNA target (yellow). (B) Unproductive base pairing within a sgRNA inhibits RNP-DNA complex formation.

Figure 2.. BLADE SELEX

(A) A vast sgRNA library contains fixed regions (black) for primer binding and a central 60-nucleotide randomized region biased toward the S. pyogenes wild-type tracrRNA sequence (various colors). (B) The sgRNA library competes in tests of Cas9 binding, target DNA binding, and DNA cleavage. (C) Successfully cleaved DNAs are tagged for selective capture of bound Cas9-sgRNA RNPs. (D) Recovered sgRNAs are amplified to regenerate an enriched sgRNA library. Rounds of selection enhance enrichment of successful sgRNAs. (E) Libraries are sequenced to determine which sgRNA sequences are successful and warrant validation.