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2.
Nat Biotechnol ; 40(2): 189-193, 2022 02.
Article in English | MEDLINE | ID: mdl-33927418

ABSTRACT

Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Zebrafish , Animals , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing , HEK293 Cells , Humans , RNA, Guide, Kinetoplastida/genetics , Ribonucleoproteins/genetics , Zebrafish/genetics
3.
Nat Biotechnol ; 39(1): 41-46, 2021 01.
Article in English | MEDLINE | ID: mdl-32690971

ABSTRACT

CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications1-4 but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities5,6. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.


Subject(s)
CRISPR-Cas Systems/genetics , Cytosine/metabolism , Gene Editing/methods , Cytidine Deaminase/metabolism , DNA/genetics , DNA/metabolism , Guanine/metabolism , HEK293 Cells , Humans
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