The Acidaminococcus sp. Cas12a nuclease recognizes GTTV and GCTV as non-canonical PAMs.
Acidaminococcus
/ enzymology
Bacterial Proteins
/ genetics
CRISPR-Associated Proteins
/ genetics
CRISPR-Cas Systems
Catalytic Domain
DNA Cleavage
Endodeoxyribonucleases
/ genetics
Endonucleases
/ genetics
Escherichia coli
/ genetics
Gene Editing
HEK293 Cells
Humans
Nucleotide Motifs
Plasmids
/ genetics
CRISPR-Cas systems
Cas nuclease
Cpf1
PAM
TIDE
TXTL
Journal
FEMS microbiology letters
ISSN: 1574-6968
Titre abrégé: FEMS Microbiol Lett
Pays: England
ID NLM: 7705721
Informations de publication
Date de publication:
01 04 2019
01 04 2019
Historique:
received:
23
12
2018
accepted:
19
04
2019
pubmed:
21
4
2019
medline:
4
6
2020
entrez:
21
4
2019
Statut:
ppublish
Résumé
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.
Identifiants
pubmed: 31004485
pii: 5475644
doi: 10.1093/femsle/fnz085
pmc: PMC6604746
pii:
doi:
Substances chimiques
Bacterial Proteins
0
CRISPR-Associated Proteins
0
Cas12a protein
EC 3.1.-
Endodeoxyribonucleases
EC 3.1.-
Endonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NIGMS NIH HHS
ID : R35 GM119561
Pays : United States
Informations de copyright
© FEMS 2019.
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