Douglas J. Dellinger

Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.

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.

Chemical and Biochemical Studies with Dithioate DNA

Abstract Dithioate DNA was synthesized and used for various biochemical studies. Results from these studies indicate that dithioate DNA is a potent inhibitor of HIV Reverse Transcriptase, activates endogenous RNase H in Hela cell nuclear extracts, and is a useful probe for studying protein-DNA interactions.

Synthesis and Biological Activity of Phosphonocarboxylate DNA

Oligodeoxynucleotides containing internucleotide phosphonoacetate esters are taken up irreversibly by cells in culture in the absence of cationic lipids. These oligonucleotides also are active in stimulating RNase H and are stable toward nucleases.

Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs

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.

Oligodeoxyribonucleotide Analogs Functionalized with Phosphonoacetate and Thiophosphonoacetate Diesters

Oligodeoxyribonucleotides with phosphonoacetate or thiophosphonoacetate internucleotide linkages can be made in high yield by solid‐phase synthesis and possess many advantages. They are highly stable to nucleases, water‐soluble, and anionic at neutral pH. They form stable duplexes with DNA and RNA, and stimulate RNase H degradation of complementary RNA. The preparation of the N,N‐(diisopropylamino)phosphinyl acetate monomers from standard protected nucleosides is described here, followed by the synthesis of phosphonoacetate and thiophosphonoate oligodeoxyribonucleotides, as well as chimeric oligomers that have these modified linkages in combination with natural or phosphorothioate linkages. Purification and characterization of these oligomers is also presented.

An electrospray mass spectrometric method for accurate mass determination of highly acid-sensitive phosphoramidites

An accurate mass determination method utilizing electrospray ionization mass spectrometry is described for analysis of several different types of phosphoramidites that are extremely acid-sensitive compounds. An earlier method, which applied a LiCl/acetonitrile system, was extended for this special application by using polymeric standards including poly(ethylene glycol) (PEG), poly(ethylene glycol) dimethyl ether (PDE) and poly(propylene glycol) (PPG). Concentrations of standards, samples and LiCl were optimized and potential impurities that affect the analyses were also investigated.

Accurate mass analysis of phosphoramidites by electrospray mass spectrometry.

A method of accurate mass determination of phosphoramidites is described. The commonly used methanol/water/acid system was replaced by LiCl-containing acetonitrile and the concentrations of LiCl, poly(ethylene glycol), and phosphoramidite samples were optimized.

Phosphoramidites as Synthons for Polynucleotide Synthesis

Abstract Methods for the rapid synthesis of DNA and RNA are described. The procedures involve using nucleoside phosphoramidites as synthons and silica as a polymeric support. Additionally, a novel reaction involving nucleoside O-alkyl methylphosphonothioates is described.

A NEW METHOD FOR SYNTHESIZING RNA ON SILICA SUPPORTS

Until recently we perceived that there was only a minimal need for methods for chemical synthesis of RNA. This was because almost any RNA sequence could be synthesized using either the SP6 or T7 promoter and an appropriate DNA duplex (1,2). However, several recent developments primarily centered on studies of intron splicing mechanisms (3–6) have led us to initiate a program in this area. We now feel that eventually new chemical methods for synthesizing RNA will be very important as an aid to helping us understand a large number of biochemical processes such as mRNA and tRNA maturation (3–6), conformational analysis of RNA (7), and the mechanisms whereby proteins recognize and bind to RNAs (8). For example, we will probably want to construct mRNA, lariat and branched tetranucleotide structures, tRNAs, viroids, 5S RNA or even ribosomal RNA in such a way that sugar or base analogues are inserted at certain key sites so that their biochemical activity can be studied. We would envisage such a synthesis to involve first preparing the analogue portion of the RNA chemically and the remainder from DNA and either the T7 or SP6 promoter. These segments would then be joined with T4-RNA ligase (1) to form the RNA of interest.