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Very Fast CRISPR using Caged Deoxythymidine

A very fast CRISPR method employing caged RNA (vfCRISPR) allows Cas9 to bind DNA without cleaving the targeted DNA until activated by light.

Liu et al. recently designed a Cas9 CRISPR system that allows genome-editing manipulations in seconds. The key to this method is the partially chemically caged guide RNA allowing the Cas9-guide RNA complex to bind to a specific genomic location without cleavage. Following light activation, vfCRISPR creates double-strand breaks at a submicrometer resolution within seconds.

The discovery of RNA-guided DNA targeting using CRISPR-Cas9 in 1993 revolutionized modern gene editing. Precise genome-editing utilizes the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ) is error-prone. NHEJ acts simultaneously to HDR and reduces the rate of high-fidelity edits. To avoid off-target effects, CRISPR based gene editing methods need to be highly precise and specific.

Liu et al. showed that light-induced synchronized cleavage using vfCRISPR enables kinetic analysis of DNA repair. Using this method, the research group revealed how cells respond to Cas9-induced double-strand breaks (DSBs) within minutes and retain the double-strand break repair protein MRE11 after DNA ligation.

The vfCRISPR approach utilizes the Streptococcus pyogenes Cas9 (Cas9) cleavage mechanism. The protospacer adjacent motif (PAM) is a 9 to 10 base pair region of the guide RNA (gRNA) that governs Cas9 binding to its target DNA. However, additional base pairing at the PAM-distal region, approximately 10 to 20 base pairs in length, is required for cleavage.

The presence of mismatches in the PAM-distal region prevents full unwinding of target DNA and conformational changes of the HNH domain required for cleavage. Based on the CRISPR system's mechanics, the researchers replaced two to three uracils at the PAM-distal region of crRNA with light-sensitive, 6-nitropiperonyloxymethyl (NPOM)–modified deoxythymidine caged nucleotides. Hybridization of crRNA to wild-type transactivating CRISPR RNA (tracrRNA) resulted in a caged guide RNA (cgRNA).

TheCas9/cgRNA complex can bind its target DNA but cannot cleave because the steric hindrance of the caging groups prevents full DNA unwinding and nuclease activation. Stimulation with light at 365 or 405 nm removes the caging groups. The pre-bound, now-activated Cas9/cgRNA complex rapidly cleaves target DNA.

The HNH domain contains approximately 30 amino acids with two conserved histidines and one asparagine. For more detail, please review the structure of the HNH homing endonuclease I-Hmul PDB ID 1U3E.

Photocaging is an attractive approach for the control and study of complex biological processes. The term “caging” is used to describe the attachment of a photolabile protecting group to a biologically active molecule. When placed at specific locations of the selected molecule, the biological function is blocked, and the molecule is inactivated. Uncaging restores the biological function. Photoremovable protecting groups allow spatial and temporal control over the release of various biologic active molecules. Examples are ATP, neurotransmitters and cell-signaling molecules, acids, bases, calcium ions, oxidants, insecticides, pheromones, and many others. Liu et al. used the light-sensitive, 6-nitropiperonyloxymethyl (NPOM)–modified deoxynucleotide thymine (dT) caged nucleotide for vfCRISPR. The structure of the NPOM modified dT and its uncaging reaction is illustrated in figure 1.

Figure 1: NPOM modified dT. The presence of the photolabile 6-nitropiperonyloxymethyl (NPOM) group at the N3 position of thymidine inhibits base-pairing and prevents hybridization. Caging refers to the attachment of a photolabile protecting group to a biologically active molecule, such as an oligonucleotide. Irradiation of the caged compound using light at the wavelength required to remove the protecting group ‘uncages” the caged compound and restores its biological function. Caged nucleoside phosphoramidites allow the synthesis of caged oligonucleotides. Caged oligonucleotides are versatile tools for PCR, the study of polymerase activity, antisense and gene silencing, regulation of restriction endonuclease activity, enzyme-free mutagenesis, and activation and deactivation of DNAzyme, as well as photochemical control of DNA function or regulation. Placing NPOM-caged dT in oligonucleotides at every five or six bases inhibit hybridization to their complementary strands. However, incorporating a single NPOM-dT residue into an oligonucleotide may not be enough to inhibit hybridization. Photo-uncaging is carried out with UV light at 365 nm for seconds or minutes. A UV transilluminator, a hand-held UV light, or a fluorescence microscope allows the uncaging of the oligonucleotide in a specific location within a cell.

Figure 2: Illustration of Cas9 activation (Modified after Liu et al. 2020). The caged guide RNA hybridizes to the seed region in the Cas9-cgRNA complex, where the caged part of the guide creates a roadblock preventing the cleavage of the DNA strand. Stimulation with UV light at 365 or 406 nm removes the caging groups and activates the Cas9-cgRNA complex. Once activated, the Cas9-cgRNA complex cleaves the DNA strand within seconds.

According to Liu et al. vfCRISPR, combined with time-resolved biochemical, sequencing, and imaging readouts allow systematic studies of DNA damage responses. The research group suggests that the combination of vfCRISPR with subcellular photoactivation will allow precise genome editing with single-allele specificity and the elimination of off-target activity. 


Reference

A general design for caging groups

Petr Klán, Tomᚊolomek, Christian G. Bochet, Aurélien Blanc, Richard Givens, Marina Rubina, Vladimir Popik, Alexey Kostikov, and Jakob Wirz; Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy. Chemical Reviews 2013, 113, 119-191. [PMC]

Lander ES; The Heroes of CRISPR. Cell. 2016;164(1-2):18-28. [Pubmed]

Yang Liu, Roger S. Zou, Shuaixin He, Yuta Nihongaki, Xiaoguang Li, Shiva Razavi, Bin Wu,Taekjip Ha; Very fast CRISPR on demand. Science  12 Jun 2020:Vol. 368, Issue 6496, pp. 1265-1269. DOI: 10.1126/science.aay8204. [Science]

MRE11 protein

NPOM dT phosphoramidite

Stracker TH, Petrini JH. The MRE11 complex: starting from the ends. Nat Rev Mol Cell Biol. 2011 Feb;12(2):90-103. [PMC]

Zuo, Z., Liu, J. Structure and Dynamics of Cas9 HNH Domain Catalytic State. Sci Rep 7, 17271 (2017). [Scientific Reports


Bio-Synthesis Inc. is pleased to offer a large variety of bioconjugates, oligonucleotides and peptides, modified and unmodified, for a number of research applications, including COVID 19 testing, CRISPR Cas9, bioanalysis and vaccine development!

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