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Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1
It is shown here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro.
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
E engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution.
Search-and-replace genome editing without double-strand breaks or donor DNA
A new DNA-editing technique called prime editing offers improved versatility and efficiency with reduced byproducts compared with existing techniques, and shows potential for correcting disease-associated mutations.
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
Adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA are described and a transfer RNA adenosine deaminase is evolved to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant.
A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog.
High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
- V. Pattanayak, Steven Lin, J. Guilinger, E. Ma, J. Doudna, David R. Liu
- BiologyNature Biotechnology
- 11 August 2013
In vitro selection and high-throughput sequencing results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence, and suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more- active guide RNA.
Opposing Effects of Tcf3 and Tcf1 Control Wnt-Stimulation of Embryonic Stem Cell Self Renewal
It is shown that an antagonistic relationship between Wnt3a and Tcf3 on gene expression regulates ESC self-renewal, and the molecular link between the effects of Wnt and the regulation of the Oct4/Sox2/Nanog network is elucidates.
Evolved Cas9 variants with broad PAM compatibility and high DNA specificity
A key limitation of the use of the CRISPR–Cas9 system for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most…
Base editing: precision chemistry on the genome and transcriptome of living cells
A comprehensive account of the state of the art of base editing of DNA and RNA is provided, including the progressive improvements to methodologies, understanding and avoiding unintended edits, cellular and organismal delivery of editing reagents and diverse applications in research and therapeutic settings.
Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification
In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases', recently engineered variants that cleave only one DNA strand per monomer.