Selectivity and Efficiency of the Ligation of The Pyrene:Abasic Base Pair by T4 DNA Ligase: Towards Abasic Lesion Detection by Ligation

Park, Hansol; Gibbs, Julianne M.

doi:10.26434/chemrxiv-2022-f529m

Cited by 1 publication

(1 citation statement)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When a double-clicked sgRNA without stabilizing modifications was used, sgRNA 0 5′ -0 3′ , we did not observe any change in the GFP expression (Figure C). Our previous work found that our double-clicked sgRNA were active in in vitro cutting assays against HPRT and other targets, including Siglec-3, indicating that the double-clicked guides could be used to target different genes. Additionally, as mentioned, with Cas-9-expressing cells we also observed INDELS formation in two targeted genes (EMX1 and WAS) using the corresponding double-clicked sgRNA without stabilizing modifications .…”

Section: Resultsmentioning

confidence: 87%

Disrupting Protein Expression with Double-Clicked sgRNA–Cas9 Complexes: A Modular Approach to CRISPR Gene Editing

Tijaro-Bulla,

Osman,

St Laurent

et al. 2023

ACS Chem. Biol.

View full text Add to dashboard Cite

CRISPR-Cas9 is currently the most versatile technique to perform gene editing in living organisms. In this approach, the Cas9 endonuclease is guided toward its DNA target sequence by the guide RNA (gRNA). Chemical synthesis of a functional single gRNA (sgRNA) is nontrivial because of the length of the RNA strand. Recently we demonstrated that a sgRNA can be stitched together from three smaller fragments through a copper-catalyzed azide–alkyne cycloaddition, making the process highly modular. Here we further advance this approach by leveraging this modulator platform by incorporating chemically modified nucleotides at both ends of the modular sgRNA to increase resistance against ribonucleases. Modified nucleotides consisted of a 2′-O-Me group and a phosphorothioate backbone in varying number at both the 5′- and 3′-ends of the sgRNA. It was observed that three modified nucleotides at both ends of the sgRNA significantly increased the success of Cas9 in knocking out a gene of interest. Using these chemically stabilized sgRNAs facilitates multigene editing at the protein level, as demonstrated by successful knockout of both Siglec-3 and Siglec-7 using two fluorophores in conjunction with fluorescence-activated cell sorting. These results demonstrate the versatility of this modular platform for assembling sgRNAs from small, chemically modified strands to simultaneously disrupt the gene expression of two proteins.

show abstract

Section: Resultsmentioning

confidence: 87%