Enhanced Cas12a editing in mammalian cells and zebrafish

Abstract Type V CRISPR–Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We sho...

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Bibliographic Details
Published in:Nucleic acids research 2019-05, Vol.47 (8), p.4169-4180
Main Authors: Liu, Pengpeng, Luk, Kevin, Shin, Masahiro, Idrizi, Feston, Kwok, Samantha, Roscoe, Benjamin, Mintzer, Esther, Suresh, Sneha, Morrison, Kyle, Frazão, Josias B, Bolukbasi, Mehmet Fatih, Ponnienselvan, Karthikeyan, Luban, Jeremy, Zhu, Lihua Julie, Lawson, Nathan D, Wolfe, Scot A
Format: Article
Language:English
Subjects:
Animals
Base Sequence
CRISPR-Cas Systems
DNA (Cytosine-5-)-Methyltransferase 1 - genetics
DNA (Cytosine-5-)-Methyltransferase 1 - metabolism
Embryo, Nonmammalian
Endonucleases - genetics
Endonucleases - metabolism
Gene Editing - methods
HEK293 Cells
HeLa Cells
Homeodomain Proteins - genetics
Homeodomain Proteins - metabolism
Humans
Inverted Repeat Sequences
Jurkat Cells
K562 Cells
Nuclear Localization Signals
Nucleic Acid Conformation
Nucleic Acid Enzymes
Plasmids - chemistry
Plasmids - metabolism
Ribonucleoproteins - genetics
Ribonucleoproteins - metabolism
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Guide, CRISPR-Cas Systems - metabolism
Transcription Factors - genetics
Transcription Factors - metabolism
Transfection
Zebrafish
Zebrafish Proteins - genetics
Zebrafish Proteins - metabolism
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Summary:Abstract Type V CRISPR–Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkz184