Versatile 3′ Functionalization of CRISPR Single Guide RNA

Specific applications of CRISPR/Cas genome editing systems benefit from chemical modifications of the sgRNA. Herein we describe a versatile and efficient strategy for functionalization of the 3′-end of a sgRNA. An exemplary collection of six chemically modified sgRNAs was prepared containing crosslinkers, a fluorophore and biotin. Modification of the sgRNA 3′-end was broadly tolerated by Streptococcus pyogenes Cas9 in an in vitro DNA cleavage assay. The 3′-biotinylated sgRNA was used as an affinity reagent to identify IGF2BP1, YB1 and hnRNP K as sgRNA-binding proteins present in HEK293T cells. Overall, the modification strategy presented here has the potential to expand on current applications of CRISPR/Cas systems.