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.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , CRISPR-Associated Proteins/chemistry , CRISPR-Cas Systems , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Gene Editing , Ribose/metabolism , Bacterial Proteins/chemistry , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/metabolism , Endodeoxyribonucleases/chemistry , HEK293 Cells , Humans , Nucleic Acids , Pathology, Molecular/methods , RNA , RNA, Guide, Kinetoplastida/genetics , Ribose/chemistryABSTRACT
We report on the synthesis of siRNAs containing both 2'-5'- and 3'-5'-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2'-5' linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5'-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5'p-rN1-(2'-5')-N2 unit in the antisense strand does not alter the 'clover leaf' bend and sugar puckers that are critical for anchoring the 5'-phosphate to Ago 2 MID domain. Importantly, 2'-5'-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2'-5'/3'-5'-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.
Subject(s)
RNA Interference , RNA, Small Interfering/chemistry , Argonaute Proteins/metabolism , Carbohydrate Conformation , HeLa Cells , Humans , Luciferases, Firefly/genetics , Molecular Dynamics Simulation , Nucleic Acid Conformation , RNA, Small Interfering/chemical synthesis , RNA, Small Interfering/immunology , Tumor Suppressor Protein p53/geneticsABSTRACT
CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar-phosphate backbone of Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA 'seed' region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2'-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2'-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2'-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.
Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , RNA, Bacterial/chemistry , DNA Cleavage , DNA, A-Form/chemistry , RNA, Bacterial/metabolism , Ribonucleoproteins/metabolism , Streptococcus pyogenes/enzymology , Streptococcus pyogenes/genetics , Structure-Activity RelationshipABSTRACT
DNA substitutions in RNA can probe the importance of A-form structure, 2'-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. Using this approach, we found that Cas9 biochemical activity tolerated significant substitution with DNA nucleotides in the clustered regularly interspaced short palindromic repeat RNA (crRNA). Only minimal RNA content was needed in or near the seed region. Simultaneous substitution at all positions with predicted crRNA-Cas9 2'-hydroxyl contacts had no effect on enzyme activity. The trans-activating crRNA (tracrRNA) also tolerated >50% substitution with DNA. DNA substitutions in the tracrRNA-pairing region of crRNA consistently enhanced cleavage activity while maintaining or improving target specificity. Together, results point to a prominent role for guide:target A-form-like helical structure and a possible regulatory role for the crRNA-tracrRNA pairing motif. A model chimeric crRNA with high activity did not significantly alter RNP assembly or target binding but did reduce Cas9 ribonucleoprotein stability, suggesting effects through conformation or dynamics. Cas9 directed by chimeric RNA-DNA guides may represent a cost-effective synthetic or molecular biology tool for robust and specific DNA cleavage.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Endonucleases/chemistry , Endonucleases/genetics , Bacterial Proteins/metabolism , CRISPR-Associated Protein 9 , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , DNA/genetics , DNA Cleavage , Endonucleases/metabolism , RNA, Bacterial/chemistry , RNA, Guide, KinetoplastidaABSTRACT
The spinach family of RNA aptamers are RNA mimics of green fluorescent protein (GFP) that have previously been designed to address the challenges of imaging RNA inside living cells. However, relatively low levels of free intracellular magnesium limited the practical use of these aptamers. Recent cell-based selections identified the broccoli RNA aptamer, which requires less magnesium for fluorescence, but the basis for magnesium preference remained unclear. Here, we find that the broccoli RNA structure is very similar to that of baby spinach, a truncated version of the spinach aptamer. Differences in stability and metal ion preferences between these two aptamers, and among broccoli mutants, are primarily associated with the sequence and structure of predicted quadruplex-flanking stem structures. Mutation of purine-purine pairs in broccoli at the terminal stem-quadruplex transition caused reversion of broccoli to a higher magnesium dependence. Unique duplex-to-quadruplex transitions in GFP-mimic RNAs likely explain their sensitivity to magnesium for stability and fluorescence. Thus, optimizations designed to improve aptamers should take into consideration the role of metal ions in stabilizing the transitions and interactions between independently folding RNA structural motifs.