DNA and RNA editing without sequence limitation using the flap endonuclease 1 guided by hairpin DNA probes

Abstract

Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.

A novel strategy for orthogonal genetic regulation on different RNA targeted loci simultaneously

“…Then FEN1 participates in removing RNA primers. Then in 2020, we used the characteristic of FEN1 to build a h air p in DNA probe s tructure- g uided n uclease system (designated the HpSGN system hereafter) [ 18 ]. The HpSGN system comprises FEN1 nuclease and hairpin DNA probe (designated hpDNA below).…”

“…The HpSGN system comprises FEN1 nuclease and hairpin DNA probe (designated hpDNA below). Significantly, a notable feature is that the cleavage is invalid when the base at the position of 1 on the target mismatches the hpDNA [ 18 ]. We inferred that this feature could be taken to build an interference platform.…”

AntiV‐SGN: a universal antiviral strategy to combat both RNA and DNA viruses by destroying their nucleic acids without sequence limitation

“…The plasmids were constructed as described before (Tian et al ., 2020). For HBV relative experiments, NLS was added to the N‐terminus of A. fulgidus and then subcloned to pcDNA3.1(+) to generate pcDNA3.1(+)‐HA‐NLS‐ A. fulgidus FEN1.…”

“…Recently, we developed and reported a hairpin DNA probe‐SGN (HpSGN) system (Tian et al ., 2020). The HpSGN system is composed of the FEN1 nuclease and the hairpin DNA probe (designated hpDNA below).…”

“…The HpSGN system is composed of the FEN1 nuclease and the hairpin DNA probe (designated hpDNA below). Moreover, the HpSGN system has been proved to mediate genomic DNA modification and mRNA degradation (Tian et al ., 2020). The HpSGN system has advantages of (i) without sequence limitation like PAM or PFS; (ii) being able to cleave both DNA and RNA simultaneously; (iii) with smaller molecular weight and size (35 kDa, 337 amino acids) to delivery in vivo and ex vivo .…”