RNA-Independent DNA Cleavage Activities of Cas9 and Cas12a
| Autor: | Hari Priya Parameshwaran, S. D. Yogesha, Mark Walter Keilbarth, Ramya Sundaresan, Rakhi Rajan |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Francisella tularensis novicida Time Factors CRISPR-Associated Proteins SpyCas9 General Biochemistry Genetics and Molecular Biology Article Substrate Specificity 03 medical and health sciences chemistry.chemical_compound Plasmid Cpf1 CRISPR-Associated Protein 9 Catalytic Domain CRISPR Deoxyribonuclease I Trypsin Cas9 endonucleases Guide RNA DNA Cleavage lcsh:QH301-705.5 Manganese Base Sequence Cas12a Chemistry Cas9 RNA DNA RNA-independent DNA cleavage Protospacer adjacent motif Kinetics 030104 developmental biology Biochemistry lcsh:Biology (General) FnoCas9 Proteolysis FnoCas12a Nucleic Acid Conformation Mobile genetic elements Mn2+-specific CRISPR activity |
| Zdroj: | Cell Reports, Vol 21, Iss 13, Pp 3728-3739 (2017) Cell reports |
| ISSN: | 2211-1247 |
| Popis: | SUMMARY CRISPR-Cas systems provide bacteria and archaea with sequence-specific protection against invading mobile genetic elements. In the presence of divalent metal ions, Cas9 and Cas12a (formerly Cpf1) proteins target and cleave DNA that is complementary to a cognate guide RNA. The recognition of a protospacer adjacent motif (PAM) sequence in the target DNA by Cas9 and Cas12a is essential for cleavage. This RNA-guided DNA targeting is widely used for gene-editing methods. Here, we show that Francisella tularensis novicida (Fno) Cas12a, FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9) cleave DNA without a guide RNA in the presence of Mn2+ ions. Substrate requirements for the RNA-independent activity vary. FnoCas9 preferentially nicks double-stranded plasmid, SpyCas9 degrades single-stranded plasmid, and FnoCas12a cleaves both substrates. These observations suggest that the identities and levels of intracellular metals, along with the Cas9/Cas12a ortholog employed, could have significant impacts in genome editing applications In Brief CRISPR-mediated gene editing involves DNA targeting using complementary guide RNAs (gRNAs). Sundaresan et al. find that Cas9/Cas12a orthologs cause RNA-independent, non-sequence-specific DNA cleavage in the presence of Mn2+ ions. These observations suggest that the type of Cas9/Cas12a and levels of intracellular metal ions may affect CRISPR-based genome editing. |
| Databáze: | OpenAIRE |
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