Abstract
New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering.
Highlights
CRISPR-Cas systems are a group of RNA-guided machineries that defend their prokaryotic hosts against invasive genetic elements in a programmable manner [1,2]
The targetable DNA-binding Cas nucleases are therein applied in generating double-stranded DNA breaks (DSBs) at specific chromosomal loci, stimulating the host repair mechanisms, including homology-directed repair (HDR) and non-homologous end joining (NHEJ), to bring about designed or error-prone genomic alterations [3]
By consuming ATP, Cas3 unwinds the dsDNA starting at the nicked site via its helicase domain to further provide ssDNA substrate for its nuclease domain, eventually leading to complete target degradation [33,34]
Summary
CRISPR-Cas systems are a group of RNA-guided machineries that defend their prokaryotic hosts against invasive genetic elements in a programmable manner [1,2]. The targetable DNA-binding Cas nucleases are therein applied in generating double-stranded DNA breaks (DSBs) at specific chromosomal loci, stimulating the host repair mechanisms, including homology-directed repair (HDR) and non-homologous end joining (NHEJ), to bring about designed or error-prone genomic alterations [3]. Such applications have been currently focused on the compact Class 2 systems with a single Cas effector on account of their simplicity and ease of heterologous use [4]. NCas can help deaminases to yield more predictable and precise genome editing compared with wild-type Cas9-based editing [8]
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