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.
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.
Phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides were prepared via a solid-phase synthesis strategy. Under Reformatsky reaction conditions, novel esterified acetic acid phosphinodiamidites were synthesized and condensed with appropriately protected 5'-O-(4, 4'-dimethoxytrityl)-2'-deoxynucleosides to yield 3'-O-phosphinoamidite reactive monomers. These synthons when activated with tetrazole were used with an automated DNA synthesizer to prepare phosphonoacetic acid modified internucleotide linkages on controlled pore glass. The phosphinoacetate coupling products were quantitatively oxidized at each step with (1S)-(+)-(10-camphorsulfonyl)oxaziridine or 3H-1,2-benzodithiol-3-one-1,1-dioxide to produce mixed sequence phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides with an average per cycle coupling efficiency of greater than 97%. Completely deprotected, modified oligodeoxynucleotides were purified by reverse-phase HPLC and characterized by ion exchange HPLC, (31)P NMR, and MALDI/TOF mass spectroscopy. Both analogues were stable toward hydrolysis with snake venom phosphodiesterase and stimulated RNase H1 activity.
An improved method for the chemical synthesis of RNA was developed utilizing a streamlined method for the preparation of phosphoramidite monomers and a single-step deprotection of the resulting oligoribonucleotide product using 1,2-diamines under anhydrous conditions. The process is compatible with most standard heterobase protection and employs a 2'-O-(1,1-dioxo-1λ(6)-thiomorpholine-4-carbothioate) as a unique 2'-hydroxyl protective group. Using this approach, it was demonstrated that the chemical synthesis of RNA can be as simple and robust as the chemical synthesis of DNA.
A novel solid-phase phosphoramidite based oligodeoxynucleotide two-step synthesis method has been developed. Keys to this method are replacement of the 5'-dimethoxytrityl blocking group with an aryloxycarbonyl and the use of N-dimethoxytrityl protection for the exocyclic amines of adenine and cytosine. With these modifications, coupling of each 2'-deoxynucleoside 3'-phosphoramidite to the growing oligodeoxynucleotide on the solid support can be followed by treatment with an aqueous mixture of peroxy anions buffered at pH 9.6. This reagent effectively removes the carbonate protecting group and simultaneously oxidizes the phosphite internucleotide linkage. As a consequence a new two-step synthesis cycle is possible. Oligodeoxynucleotides synthesized using this approach are identical to authentic samples when tested by a variety of analytical techniques.
Phosphonoformate oligodeoxyribonucleotides were prepared via a solid phase synthesis strategy. The first step in the preparation of appropriate synthons was condensation of bis(N,N-diisopropylamino)phosphine and diphenylmethylsilylethyl chloroformate in the presence of sodium metal to yield formic acid, [bis(N,N-diisopropylamino)phosphino]-beta-(diphenylmethylsilylethyl) ester. The product of this reaction was then condensed with appropriately protected 2'-deoxynucleosides using 4,5-dicyanoimidazole to yield the 3'-O-phosphinoamidite reactive monomers. The exocyclic amines of cytosine, adenine, and guanine were protected with 9-fluorenylmethyloxycarbonyl, and oligodeoxyribonucleotides were synthesized on controlled pore glass using the hydroquinone-O,O'-diacetic acid linker. Synthons were sequentially added to this support using tetrazole as an activator, oxidized to phosphonoformate, and the transient 5'-protecting group was removed with acid. Following total synthesis of an oligomer, protecting groups were removed with TEMED.HF and products purified by HPLC. These analogues were resistant to nucleases, formed duplexes with complementary RNA (A-form), and, as chimeric oligomers containing phosphate at selected sites, stimulated RNase H1 activity.
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.
INTRODUCTIONAlthough synthetic DNA having the natural 3'-5' phosphodiester linkage has proven to be extremely valuable for a large number of biochemical and biological applications,lf certain key uses require modified synthetic polynucleotides. For these appli-
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.