PT  - JOURNAL ARTICLE
AU  - Chon-Hwa Tsai-Morris
AU  - Sydney Hertafeld
AU  - Yvonne Rosario
AU  - James Iben
AU  - Eric Chang
AU  - Ling Yi
AU  - Steven L. Coon
AU  - Stephen G. Kaler
AU  - Ryan Dale
AU  - Benjamin Feldman
TI  - Detection of precisely edited CRISPR/Cas9 alleles through co-introduced restriction-fragment length polymorphisms
AID  - 10.1101/2021.04.20.440537
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.04.20.440537
4099  - http://biorxiv.org/content/early/2021/04/20/2021.04.20.440537.short
4100  - http://biorxiv.org/content/early/2021/04/20/2021.04.20.440537.full
AB  - CRISPR/Cas9 is a powerful tool for producing genomic insertions and deletions (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 single-stranded oligo deoxynucleotide (ssODN) donor templates, we pioneered a strategy of adding synonymous changes to create novel restriction-enzyme (RE) sites, allowing detection of rare precise edits in a modified fluorescent-PCR fragment assay. We have named this process TIARS (test for incorporation of added recognition sites). Aided by TIARS, we induced two distinct amino-acid substitutions (T979I and P1387S) in the atp7a gene among somatic tissues of CRISPR-Cas9-treated F0 zebrafish. One of these F0s transmitted the allele to atp7aT979I/+ F1 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.Competing Interest StatementThe authors have declared no competing interest.