Efficient genome editing by a miniature CRISPR-AsCas12f1 nuclease in Bacillus anthracis

The miniature nuclease AsCas12f1 from Acidibacillus sulfuroxidans edits Bacillus anthracis at complete efficiency, on both the chromosome and a resident plasmid. Electroporating a single plasmid carrying donor DNA, a gene-specific sgRNA and the AsCas12f1 cassette into B. anthracis A16R deleted the 1242 bp chromosomal htrA gene in all randomly selected colonies for each of two target spacers (9 of 9 and 9 of 9), shifting the diagnostic amplicon from 2.3 kb to 1.0 kb, with a similar result for deletion of lef on the pXO1 plasmid.

Editing efficiency with AsCas12f1 in B. anthracis depends sharply on donor homology arm length, with a threshold between 200 and 400 bp. Homology arms of 50 bp or 100 bp gave no htrA deletion in any of 30 randomly selected colonies, only wild-type amplicons. At 200 bp, just 2 of 15 colonies carried the pure deletion while 13 of 15 were heterozygous with both bands. At 400 bp and 800 bp, all colonies tested (15 of 15 each) carried the complete deletion.

AsCas12f1 supports excision of a multi-kilobase chromosomal region, not just single genes: the prophage lambdaBa03, whose flanking amplicon exceeds 19 kb in the parental strain, was excised in all 9 randomly selected colonies with 800 bp homology arms, yielding the expected 2.2 kb amplicon. The same 100% efficiency (9 of 9) held with homology arms reduced to 400 bp, so the arm-length requirement does not scale up with deletion size.

Of four promoters screened by driving the mScarlet reporter in B. anthracis, the PxylA promoter from Bacillus subtilis is both strictly inducible and relatively high-expressing, making it suitable for controlling a toxic endonuclease.

A two-plasmid CRISPR-AsCas12f1 system for B. anthracis separates the nuclease from the guide, enabling sequential multi-gene modification: one plasmid co-expresses AsCas12f1 and the endonuclease I-SceI under the inducible PxylA promoter, and a second plasmid, pSS-FD, carries the sgRNA cassette. Targeting htrA with this system deleted the gene in all 9 randomly selected colonies.

Inducing I-SceI expression with xylose cures the sgRNA plasmid from edited B. anthracis, clearing the way for a subsequent editing round, though only partially: 12 of 50, 6 of 50 and 17 of 50 recombinant colonies across three independent experiments lost kanamycin resistance, while no kanamycin-sensitive colony arose under non-inducing conditions after one passage.