A new family of CRISPR-type V nucleases with C-rich PAM recognition

Autor: Tomas Urbaitis, Giedrius Gasiunas, Joshua K Young, Zhenglin Hou, Sushmitha Paulraj, Egle Godliauskaite, Mantvyda M Juskeviciene, Migle Stitilyte, Monika Jasnauskaite, Megumu Mabuchi, G Brett Robb, Virginijus Siksnys
Rok vydání: 2022
Předmět:
Zdroj: EMBO reports, Hoboken : Wiley, 2022, vol. 23, iss. 12, art. no. e55481, p. [1-15]
ISSN: 1469-3178
1469-221X
Popis: Most CRISPR-type V nucleases are stimulated to cleave double-stranded (ds) DNA targets by a T-rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA-guided nuclease, Cas12l, that exclusively recognizes a C-rich (5'-CCY-3') PAM. The organization of genes within its CRISPR locus is similar to type II-B CRISPR-Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR-associated nucleases may be harnessed as genome editing reagents.
Databáze: OpenAIRE
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