Abstract
The class 2 CRISPR-Cas endonuclease Cas12a (previously known as Cpf1) offers several advantages over Cas9, including the ability to process its own array and the requirement for just a single RNA guide. These attributes make Cas12a promising for many genome engineering applications. To further expand the suite of Cas12a tools available, we tested 16 Cas12a orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cas12a), exhibited robust indel formation. We also showed that Mb3Cas12a displays some tolerance for a shortened PAM (TTN versus the canonical Cas12a PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology.
Keywords:
CRISPR-Cas; gene therapy; genome editing.
MeSH terms
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Bacterial Proteins / genetics*
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Bacterial Proteins / metabolism
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Base Pairing
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Base Sequence
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CRISPR-Associated Proteins / genetics*
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CRISPR-Associated Proteins / metabolism
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CRISPR-Cas Systems*
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Clustered Regularly Interspaced Short Palindromic Repeats*
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Endodeoxyribonucleases / genetics*
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Endodeoxyribonucleases / metabolism
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Gene Editing / methods*
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Genetic Engineering / methods
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HEK293 Cells
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High-Throughput Nucleotide Sequencing
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Humans
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INDEL Mutation
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Isoenzymes / genetics
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Isoenzymes / metabolism
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Moraxella / enzymology
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Moraxella / genetics
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Nucleic Acid Conformation
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Plasmids / chemistry
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Plasmids / metabolism
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RNA, Guide, CRISPR-Cas Systems / chemistry
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RNA, Guide, CRISPR-Cas Systems / genetics*
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RNA, Guide, CRISPR-Cas Systems / metabolism
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Sequence Alignment
Substances
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Bacterial Proteins
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CRISPR-Associated Proteins
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Isoenzymes
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RNA, Guide, CRISPR-Cas Systems
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Cas12a protein
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Endodeoxyribonucleases