Engineered CRISPR prime editors with compact, untethered reverse transcriptases

The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use th...

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Bibliographic Details
Published in:Nature biotechnology 2023-03, Vol.41 (3), p.337-343
Main Authors: Grünewald, Julian, Miller, Bret R., Szalay, Regan N., Cabeceiras, Peter K., Woodilla, Christopher J., Holtz, Eliza Jane B., Petri, Karl, Joung, J. Keith
Format: Article
Language:English
Subjects:
631/61/201
631/61/338
Agriculture
Animals
Bioinformatics
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Brief Communication
C-Terminus
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonuclease I - genetics
Editing
Gene Editing - methods
Humans
Leukemia
Life Sciences
Medical research
Mice
Moloney murine leukemia virus - genetics
Mutation
Proteins
RNA-directed DNA polymerase
RNA-Directed DNA Polymerase - genetics
Streptococcus pyogenes - genetics
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Summary:The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing. A split prime editor architecture facilitates screening and engineering of improved variants.
ISSN:1087-0156
1546-1696
DOI:10.1038/s41587-022-01473-1