Abstract
CRISPR systems build adaptive immunity against mobile genetic elements by DNA capture and integration catalysed by Cas1–Cas2 protein complexes. Recent studies suggested that CRISPR repeats and adaptation module originated from a novel type of DNA transposons called casposons. Casposons encode a Cas1 homologue called casposase that alone integrates into target molecules single and double-stranded DNA containing terminal inverted repeats (TIRs) from casposons. A recent study showed Methanosarcina mazei casposase is able to integrate random DNA oligonucleotides, followed up in this work using Acidoprofundum boonei casposase, from which we also observe promiscuous substrate integration. Here we first show that the substrate flexibility of Acidoprofundum boonei casposase extends to random integration of DNA without TIRs, including integration of a functional gene. We then used this to investigate targeting of the casposase-catalysed DNA integration reactions to specific DNA sites that would allow insertion of defined DNA payloads. Casposase–Cas9 fusions were engineered that were catalytically proficient in vitro and generated RNA-guided DNA integration products from short synthetic DNA or a gene, with or without TIRs. However, DNA integration could still occur unguided due to the competing background activity of the casposase moiety. Expression of Casposase-dCas9 in Escherichia coli cells effectively targeted chromosomal and plasmid lacZ revealed by reduced β-galactosidase activity but DNA integration was not detected. The promiscuous substrate integration properties of casposases make them potential DNA insertion tools. The Casposase–dCas9 fusion protein may serves as a prototype for development in genetic editing for DNA insertion that is independent of homology-directed DNA repair.
Highlights
Horizontal gene transfer between prokaryotic cells is a major driving force for their evolution but is resisted by host self-defence proteins that have evolved to maintain the genetic status quo [1,2,3]
Purified A. boonei casposase (Supplementary Figure S2A) catalysed disintegration of a DNA fork (Fork-3) more effectively in manganese compared with magnesium
LE30-top was not integrated at all and ssran19 was a poor ssDNA substrate – each lacks a cytosine at the -3 position from the 3 end, a nucleotide that is important for catalysis by A. boonei casposase (Figure 1B lanes 3, 6 and Figure 1C) [15,18]
Summary
Horizontal gene transfer between prokaryotic cells is a major driving force for their evolution but is resisted by host self-defence proteins that have evolved to maintain the genetic status quo [1,2,3]. CRISPR–Cas systems provide adaptive immune defence in bacteria and archaea [4,5,6]. CRISPR–Cas systems centre on specialised chromosomal sites comprising a CRISPR DNA array and cas (CRISPR-associated) genes. The CRISPR locus is a depot of 25–40 base pair DNA ‘spacer’ fragments acquired in cells during previous encounters with DNA from mobile genetic elements (MGEs). The spacer sequences in CRISPRs alternate with DNA ‘repeats’ that provide sequence and structural elements necessary for correct functioning of the CRISPR locus. A crRNA can be targeted to MGE DNA by base pairing catalysed by Cas ‘Interference’ protein complexes, which include nucleases to destroy MGE DNA, mounting an immunity-based defence
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