Miniseq

DNA from the PAM screen was prepared for deep sequencing by first amplifying a 135-bp region from the reporter plasmid isolated from IPTG-induced and uninduced cultures grown in M9 xylose medium. These oligos that contained the necessary adapters for analysis by Illumina MiniSeq are prDC739 and prDC740. Once amplified, the DNA fragments were purified with AMPure XP magnetic beads (Beckman Coulter). A 0.9× bead purification was used followed by two 80% ethanol washes. After washing, the beads were dried and the DNA was eluted in twice the original volume with nuclease-free water. These amplified DNA fragments were then indexed, and phase-shifting staggers were inserted downstream of the Illumina sequencing primer site using an 8-cycle PCR (prDC731 to prDC738). Nextera indices 501, 502, 503, 504, 701, 702, 703, and 704 were used in this work, and staggers were added to increase the diversity. Once amplified, the PCR products were purified with AMPure XP magnetic beads similarly to the first PCR, but instead eluting in the original PCR volume. These samples were quantified on a NanoDrop 2000c spectrophotometer and by agarose gel electrophoresis. The samples were then quantified on a QuBit, analyzed with the Bioanalyzer HS DNA chip, and pooled. Samples were run on the MiniSeq Mid Output Kit with 300 cycles (150-bp paired end) with 20% PhiX spike-in on an Illumina MiniSeq.

The target sites were amplified by a total of three rounds of PCR and subjected to the Illumina MiniSeq or iSeq 100 sequencing system as previously described.²⁹ (link) Briefly, 3 μL of cell lysate or 1 μL of isolated genomic DNA was subjected to the first round of PCR, and then,1 μL of the first PCR product was used in the second round of PCR. The Illumina TruSeq HT dual index adaptor sequences were attached with index PCR primer pairs using 1 μL of second-round PCR product. The size of the PCR amplicon was confirmed in 2% agarose gel and the amplicons were subjected to 150-bp paired-end sequencing using the Illumina MiniSeq or iSeq 100 sequencing system. The paired-end reads were joined using the fastq-join tool (https://github.com/brwnj/fastq-join). Targeted deep sequencing analysis was performed using MAUND³⁰ (link) (https://github.com/ibs-cge/maund), and all results were confirmed by Cas-Analyzer (http://www.rgenome.net/cas-analyzer/).³¹ (link) Substitution and indel frequencies were quantified as the percentage of total sequencing reads, and the threshold of editing activity was set to above 0.5%.