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

Eman A Ageely,Philip J Jensik,Masad J Damha,Leonora Abdullahu,Keith T Gagnon,Daniel O’Reilly,Ramadevi Chilamkurthy,Sunit Jana

doi:10.1038/s41467-021-26989-z

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.

Highlights

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics
Acidominococcus species Cas12a (AsCas12a) and a 39nucleotide CRISPR RNAs (crRNAs) were used for this study
The primary question was whether the pseudoknot structure was too complex or sensitive for efficient chemical modification, which previous studies had suggested[74,78,81]

Summary

Introduction

CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. Cas[9] from Streptococcus pyogenes (SpCas9) and Cas12a, known as Cpf[1], from Acidominococcus species (AsCas12a) are among the most heavily co-opted and engineered CRISPR-Cas systems for creation of model organisms and development of human therapeutics[22,23,24,25,26,27,28,29,30,31,32,33] They both efficiently induce DNA double-strand breaks and guide genome editing in mammalian cells[16,22,23,33,34,35,36,37,38], utilize a single Cas enzyme and can function with a single-guide RNA (sgRNA), Cas[9] functions as a dual-guide RNA in nature[8,16,23,33]. It appears that cis and trans activity cannot be fully uncoupled for recognition of double-stranded targets via trans activity without cis cleavage of the target strand[45,46]

Methods

Results

Conclusion