CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single ...guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.
Abstract
CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly ...matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.
RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal ...for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3'-CH2-CO-NH-5' amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P-OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5'-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications.
CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, ...tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2′-O-methyl-3′-phosphonoacetate (MP) modifications can be substantially more effective than 2′-O-methyl-3′-phosphorothioate (MS) modifications at the 3′ ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3′ ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3′ ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3′ ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3′ ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3′ ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.
RNA sequences having up to three consecutive internal amide linkages were synthesized and studied using UV and NMR spectroscopy. The amide modifications did not interfere with normal base-pairing and ...A-type RNA conformation. Three consecutive amides were well tolerated in the passenger strand of siRNA and caused little change in RNAi activity.
The protective influence of social relationships on health is well documented; however, not all relationships are positive and negative aspects of relationships may be detrimental. Relatively less is ...known about the relationships characterized by both positivity and negativity (i.e., ambivalence). The goal of this study was to examine the relative influence of ambivalence in relationship quality and social behavior on cardiovascular response.
104 healthy young adults were randomly assigned to bring in either a supportive or ambivalent same-sex friend to the experiment. Participants were also randomly assigned to receive positive, negative, ambivalent or ambiguous feedback from their friend after giving a series of speeches. Cardiovascular response was obtained before, during, and after the social stressor (speech task).
Results indicate a significant effect of relationship type before, during, and after the stressor task. Adjusting for baseline, heart rate reactivity and anxiety was significantly higher among those assigned to ambivalent friends relative to those assigned to supportive friends during the stressor task (ps<.05). There was also a significant effect of behavioral feedback during the speech task, such that those receiving ambivalent messages had the greatest systolic blood pressure (SBP) and diastolic blood pressure (DBP) reactivity (p<.05); however, there was no interaction between relationship and feedback conditions. Those in the ambivalent friend condition also exhibited significantly higher SBP, DBP, and anxiety during the baseline and recovery periods (ps<.05).
These findings suggest that both relationship quality and the actions of relationships may have a significant influence on health-relevant physiology.
•We examine the relative influence of relational and behavioral ambivalence.•Heart rate reactivity was higher among those assigned to ambivalent friends.•Blood pressure reactivity was higher among those who received ambivalent feedback.•No interaction between friendship quality and valence of feedback on reactivity.•Blood pressure and anxiety higher during recovery for those with ambivalent friends.
Phosphorus‐modified phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides were chemically synthesized and their biochemical properties evaluated. Under physiological pH, these DNA ...analogs possess negative charge and form stable, complementary A‐like DNA:RNA heteroduplexes when analyzed via circular dichroism spectroscopy. Phosphonoacetate and thiophosphonoacetate oligomers were found to stimulate RNase H activity and to be completely resistant to degradation by snake venom phosphodiesterase, DNase I and HeLa cell nuclear extract. Further research has demonstrated that neutral, esterified forms of these analogs can be taken up by cells. Phosphonoacetate and thiophosphonoacetate oligomers therefore represent a new class of oligodeoxyribonucleotide analogs having phosphorus– carbon bonds with considerable potential for use in biological research.
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