Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles

Abstract

CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5′ handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2′-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2′-hydroxyl sensitivity. Modified 5′ pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5′ pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5′ pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.

Chemical Modifications of CRISPR RNAs to Improve Gene-Editing Activity and Specificity

“…More recently, Gagnon, Damha, and co-workers reported similar results that crRNAs optimized using a structure-guided approach and containing up to 13 2′-F nucleotides in the 5′-handle pseudoknot had activity similar to the unmodified crRNA . This study also showed that individual 2′-NH 2 modifications of the positions that tolerated the 2′-F nucleotides in the 5′-handle pseudoknot decreased the Cas12a activity.…”

“…Modification of the 5′-handle pseudoknot strongly decreased the activity in both in vitro and cellular assays . A more recent study also showed that DNA modifications were not tolerated in the 5′-handle pseudoknot of crRNA in an assay that used HEK293T cells stably expressing EGFP …”

Nucleoside Analogues with a Seven-Membered Sugar Ring: Synthesis and Structural Compatibility in DNA–RNA Hybrids

“…In conjunction with UV denaturation studies, they revealed that OxT 2 and OxT 3 are suitable to be incorporated in the DNA strand of hybrid duplexes due to their favorable structural and functional features, such as dihedral angle preferences and Hbond interactions. Together, they account for a rare type of "DNA-like" modification that, on the one hand, is well accommodated inside DNA−RNA hybrids, thereby with potential applications in xenobiology, 1 and antisense 30 and CRISPR technologies, 31 and, on the other hand, expands the "toolbox" of modifications that nucleic acid chemists and biologists can experiment with.…”

“…R f = 0.44, hexane/EtOAc (3:2, v/v). 31 To a solution of 13 (130 mg, 0.17 mmol) in dry CH 2 Cl 2 (1 mL) were added ( i Pr) 2 NEt (92.0 μL, 0.51 mmol) and 2-cyanoethyl N,Ndiisopropylchlorophosphoramidite (114 μL, 0.51 mmol) at room temperature. The reaction mixture was stirred at 90 °C for 2 h in a microwave sealed reactor vessel tube.…”

“…R f = 0.46, hexane/EtOAc (3:2, v/v). 31 Oligonucleotide Synthesis and Purification. All oligonucleotides (ONs) were synthesized on an Applied Biosystems (ABI) 3400 DNA synthesizer on a 1 μmol scale using UnyLinker CPG as the solid support, mesh 500 Å. DNA phosphoramidites and 2′,4′-modified phosphoramidites were prepared as 0.1 M solutions in acetonitrile and RNA phosphoramidites as 0.15 M solutions in acetonitrile.…”