From this mapping, we determined which array sequences were present in the genome. elegans genome, reads that failed to align to the genome were aligned to the sequences of all injected plasmid species. After aligning all reads to the reference C. Two strains with off target insertions of unc-119(+) were located by whole genome sequencing (Illumina). It is possible this sequence represents a de novo event segregating in this strain. These sequences document six different healing events: three chromosomes healed by NHEJ (red, blue, purple), one chromosome (green) incorporated a truncated GFP, one chromosome (brown) inserted 4 nucleotides at the cut site, and one chromosome (pink) inserted a novel segment with sequence variations (underlined). Strains 1, 2, 4, 6 and 7 likely originated from a single parent, and strains 3 and 8 likely originated from a single parent. Unique alleles are individually color coded to highlight sibling relationships. For each strain, the sequences for both chromosomes are shown. (Top) the native unc-32 target site is shown with the guide RNA binding site and Cas9 cut site highlighted. (B) Sequencing results for the two unc-32 loci in each strain. Expected amplicon sizes for the native locus and for a complete gfp + Cbr-unc-119 insert are indicated. (A) PCR amplification of the unc-32 target locus. These lines were analyzed by PCR amplification of the targeted insertion site (A) and then followed by sequencing (B).

Some lines failed to generate GFP+ animals (8/20 lines) by the time the secondary plate starved.

At starvation of the primary plates, array+, gfp- animals were singled to generate secondary plates, and the plates were incubated until starvation. (A and B) Dead-end target sites: 20 EG9881 animals were injected with a guide RNA and repair template for tagging unc-32 with gfp. S3 Fig: Unintended CRISPR outcomes: dead-end targets and dark inserts.

Api shift select unc