Detection of precisely edited CRISPR/Cas9 alleles through co-introduced restriction-fragment length polymorphisms

ABSTRACT CRISPR/Cas9 is a powerful tool for producing genomic in sertions and del etions (indels) to interrogate gene function. Modified CRISPR/Cas9 protocols can produce targeted genetic changes that are more precise than indels, but founder recovery is less efficient. Focusing on producing missense mutations in zebrafish using s ingle- s tranded o ligo d eoxy n ucleotide (ssODN) donor templates, we pioneered a strategy of adding synonymous changes to create novel r estriction- e nzyme (RE) sites, allowing detection of rare precise edits in a modified fluorescent-PCR fragment assay. We have named this process TIARS ( t est for i ncorporation of a dded r ecognition s ites). Aided by TIARS, we induced two distinct amino-acid substitutions (T979I and P1387S) in the atp7a gene among somatic tissues of CRISPR-Cas9-treated F 0 zebrafish. One of these F 0s transmitted the allele to atp7a T979I/+ F 1 progeny, and trans-heterozygosity of this allele against a null atp7a allele causes hypopigmentation, consistent with more severe pigment deficits in zebrafish or humans carrying only null mutations in atp7a/ATP7A . Design of ssODNs with novel RE recognition sites is labor-intensive, so we developed an in silico tool, TIARS Designer, and performed bioinformatic validation indicating that TIARS should be generalizable to other genes and experimental systems that employ donor template DNA.