Pseudogene-Mediated Gene Conversion After CRISPR-Cas9 Editing Demonstrated by Partial CD33 Conversion with SIGLEC22P
Pseudogene-Mediated Gene Conversion After CRISPR-Cas9 Editing Demonstrated by Partial CD33 Conversion with SIGLEC22P
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DOI：
10.1089/crispr.2021.0052
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发表时间：
2021-09-23
期刊：
影响因子：
3.7
通讯作者：
Estus, Steven
中科院分区：
文献类型：
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作者：
Shaw, Benjamin C.;Estus, Steven
尽管基因编辑工作流程通常考虑脱靶编辑的可能性，但据我们所知，假基因指导的同源性修复以前尚未报道。在此，我们采用CRISPR-Cas9策略靶向切除U937人单核细胞系中CD 33中的外显子2。通过使用已知标记由外显子2编码的IgV结构域的临床相关抗体（P67.6，吉妥珠单抗）筛选候选克隆细胞系。除了预期的外显子2缺失外，我们还发现了意想不到的P67.6阴性细胞系，其明显保留了CD 33外显子2。测序显示，这些细胞系在同源性修复过程中经历了来自附近SIGLEC 22 P假基因的基因转换，导致相对于CD 33的三个错义突变。异位表达研究证实P67.6表位依赖于这些氨基酸。总之，我们报告了假基因指导的同源修复可能导致异常的CRISPR基因编辑。
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.