Extended Data Table 1.

Oligonucleotides used in this study.

Oligonucleotide	Sequence (5′−3′)	Notes
Cse1 (forward)	GCGCGCCATGGCTAATTTGCTTATTGATAACTGGATCC	Ncol + Cse1 fragment (pRSF-Duet ORF1)
Cse1 (reverse)	GGCCCGCGGCCGCTCAGCCATTTGATGGCCCTCC	Cse1 fragment + NotI (pRSF-Duet ORF1)
Cse2-Cas7-Cas5e-Cas6e (forward)	GCGCGGGTACCAGATGGCTGATGAAATTGATGCA	Kpnl + Cse2-Cas7-Cas5e-Cas6e fragment (pET52b)
Cse2-Cas7-Cas5e-Cas6e (reverse)	CGCGCGCGGCCGCTCACAGTGGAGCCAAAGATAG	Cse2-Cas7-Cas5e-Cas6e fragment + NotI (pET-52b)
Cse2-Cas7-Cas5e-Cas6e (forward)	GCGCGCCATGGGTCATCACCACCATCATCACGGTGCACTTGAAGTCCTCTTTC	Ncol + 6XHIS + Precission (pET-52b)
R-loop mimic (target strand)	CTGTTGGCAAGCCAGGATCTGAACAATACCGTCATCGAGCACTGCACAGA
R-loop mimic (non-target strand)	TCTGTGCAGTGCTCGATGTTTTATTTAT
SUMO-Cas3 (forward)	GCGCCGCGGATCCATGGAACCTTTTAAATATATATGCCAT	BamHI + Cas3 fragment (pET28b-SUMO vector)
SUMO-Cas3 (reverse)	GCGCCGCCTCGAGTTATTTGGGATTTGCAGGGAT	Cas3 fragment + Xhol (pET28b-SUMO vector)
Target plasmid construction (non-target strand)	CATGG $\mathrm{ATG}$ACGGTATTGTTCAGATCCTGGCTTGCCAACAGCTGCA	Ncol overhang + Target sequence + Pstl overhang (pCDF-Duet1) $\mathrm{PAM}$ altered as indicated in text
Target plasmid construction (target strand)	GCTGTTGGCAAGCCAGGATCTGAACAATACCGT $\mathrm{CAT}$C	Pstl + non-target sequence + Ncol (pCDF-Duet1) $\mathrm{PAM}$ altered as indicated in text.
dsDNA1 Fluorescent ATG PAM substrate (forward)	AACTTTAATAAGGAGATATACCATGGATG	5′–6-FAM label, PCR product used in main text EMSA and Cas3 cleavage assays.
dsDNA1 Fluorescent ATG PAM substrate (reverse)	GCGGCCGCAAGCTTGTC	5′- Cy5 label, PCR product used in main text EMSA and Cas3 cleavage assays.
dsDNA2 Fluorescent substrate (forward)	TAATACGACTCACTATAGGG	T7 Forward 5′–6-FAM label. Used with dsDNA1 (reverse) primer to amplify set of 5′-ATA, ATT, ATC, ATG, AGG and CTG PAM substrates from target plasmids.
dsDNA3 (non-target strand)	AGATATACATGG $\mathrm{ATG}$ACGGTATTGTTCAGATCCTGGCTTGCCAACAGCTGCAGGTCGACA	5′ 6-FAM or HEX label, $\mathrm{PAM}$ altered (base mutation or inosine substitution) as indicated in text.
dsDNA3 (target strand)	TGTCGACCTGCAGCTGTTGGCAAGCCAGGATCTGAACAATACCGT $\mathrm{CAT}$CCATGTATATCT	$\mathrm{PAM}$ altered (base mutation or inosine substitution) as indicated in text.
crRNA expression cassette	GCGCCGGGAATTCCCTGCATTAGG $\mathrm{TAATACGACTCACTATAGG}$ $\mathrm{GAGTTCCCCGCGCCAGCGGGGATAAACCG}$ACGGTATTGTTCAGATCCTGGCTTGCCAACAG $\mathrm{GAGTTCCCCGCGCCAGCGGGGATAAACCG}$ACGGTATTGTTCAGATCCTGGCTTGCCAACAG $\mathrm{GAGTTCCCCGCGCCAGCGGGGATAAACCG}$ACGGTATTGTTCAGATCCTGGCTTGCCAACAG $\mathrm{GAGTTCCCCGCGCCAGCGGGGATAAACCG}$ACGGTATTGTTCAGATCCTGGCTTGCCAACAG $\mathrm{GAGTTCCCCGCGCCAGCGGGGATAAACCG}$ACGGTATTGTTCAGATCCTGGCTTGCCAACAG $\mathrm{GAGTTCCCCGCGCCAGCGGGGATAAACCG}$ACGGTATTGTTCAGATCCTGGCTTGCCAACAG $\mathrm{GAGTTCCCCGCGCCAGCGGGGATTTTGCC}$ $\mathrm{CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTT}$GGACCGGATCCCCG	The crRNA expression cassette containing six identical repeat-spacer units was de novo synthesized and inserted to the pHSG-398 vector. The T7 promoter, T7 terminator, and the repeats are highlighted green, red, and yellow, respectively.