design: PAM determination assay was performed incubating of 170 nM of -synCas-crRNA (targeting 23 nt downstream the 7 random nucleotides) and 100 nM of PCR fragment from the DNA library in 1 x NEB 3.1 buffer (50 mM Tris-HCl pH 7.9, 100 mM NaCl, 10 mM MgCl2, 100 g ml1 BSA) for 1 hour at 37 C. Reaction was stopped added 6X loading dye (NEB) with EDTA and run 2% agarose gel. Gels were dyed with SYBR gold (ThermoFisher) and imaged with ChemiDoc XRS + System (Bio-Rad). The longer fragment, that contained the 7 random nucleotides, was purified from the agarose gel with GeneJet Gel Extraction kit (ThermoFisher). Sequencing libraries of the purified fragments were prepared following Microplex-library-prep-v3 User Guide (version 2 15_06_2021) with the corresponding kit Microplex-library-prep-v3 (Diagenode,  Cat. # C05010001) and indexes Diagenode 24 Dual indexes for MicroPlex Kit v3 (Diagenode, Cat. # C05010003).