RNA-triggered cell killing with CRISPR–Cas12a2

Phylogenetic analysis The amino acid sequence of GeCas12a2 was aligned with other Cas12a2 nuclease sequences 15 , 59 using Clustal Omega 60 . The alignment was trimmed with ClipKIT 61 and used to reconstruct a phylogeny with IQ-TREE v2.0.3 (-m MFP -T 8 -B 1000) 62 , using a maximum-likelihood approach. The phylogeny included Cas12a orthologues and used Cas12c as an outgroup. Branch confidence was reported as ultrafast bootstrap values (ranging from 0 to 100). PFS library preparation A PFS-containing plasmid library (CBS-6873) was constructed to include a target sequence (Supplementary Tables 8 and 9 ) followed by five randomized nucleotides (NNNNN). The target sequence was placed under a PJ23119 promoter and cloned into a low-copy sc101 origin plasmid (about five copies per cell). The library was generated by amplifying a target-encoding plasmid with primers ODpr23 and ODpr24 (Supplementary Tables 9 and 10 ). The forward primer contained a 5-nt randomized overhang. The PCR product was treated with DpnI to remove template DNA, ligated, and electroporated into Escherichia coli ( E. coli ) TOP10, yielding over two million transformants (approximately 2,000-fold library coverage). PFS library depletion The PFS preference of the GeCas12a2 nuclease (CBS-6874) was assessed by targeting the CBS-6873 PFS plasmid library with a CAO1-targeting CRISPR RNA (crRNA) plasmid (CBS-6875) or a non-targeting crRNA plasmid (CBS-6876). …