Pseudogene-Mediated Gene Conversion after CRISPR-Cas9 Editing Demonstrated by Partial CD33 Conversion with SIGLEC22P

Benjamin C. Shaw, Steven Estus

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocyte cell line. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines, which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.

Original languageEnglish
Pages (from-to)699-709
Number of pages11
JournalCRISPR Journal
Issue number5
StatePublished - Oct 2021

Bibliographical note

Funding Information:
We would like to thank Yuriko Katsumata for bioinfor-matics support, Ann M. Stowe for flow cytometry support, the University of Kentucky Light Microscopy Core Facility for help with image acquisition and processing, and the University of Kentucky Flow Cytometry and Immune Monitoring Core Facility, supported in part by the Office of the Vice President for Research, the Markey Cancer Center and an NCI Center Core Support Grant (P30CA177558).

Funding Information:
This work was supported by F99NS120365 to B.C.S. from the National Institute of Neurological Disorders and Stroke and by RF1AG059717-01S1 and R21AG068370 to S.E. from the National Institute on Aging.

Publisher Copyright:
© Copyright 2021, Mary Ann Liebert, Inc., publishers 2021.

ASJC Scopus subject areas

  • Biotechnology
  • Genetics


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