Unpredicted central inversion in a sgRNA flanked by inverted repeats

Guannan Wang; Saraswati Sukumar

doi:10.1007/s11033-020-05524-1

Unpredicted central inversion in a sgRNA flanked by inverted repeats

Mol Biol Rep. 2020 Aug;47(8):6375-6378. doi: 10.1007/s11033-020-05524-1. Epub 2020 May 18.

Authors

Guannan Wang^{1

2

3}, Saraswati Sukumar^{4

5}

Affiliations

¹ Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA. gw288@georgetown.edu.
² Department of Breast and Thyroid Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, Hubei, China. gw288@georgetown.edu.
³ Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Rd NW, New Research Building, Room E512, Washington, D.C., 20007, USA. gw288@georgetown.edu.
⁴ Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA. saras@jhmi.edu.
⁵ Department of Oncology, Johns Hopkins University School of Medicine, 1650 Orleans Street, CRB1/Room 143, Baltimore, MD, 21287, USA. saras@jhmi.edu.

PMID: 32424520
DOI: 10.1007/s11033-020-05524-1

Abstract

In genome engineering, sgRNAs define the genomic target to be modified in CRISPR/Cas9 system. Either for single gene editing or genome-wide screens, sgRNAs are cloned into plasmid vectors. During our performance of CRISPR/Cas9 gene knock out, we found that the central part of a sgRNA was inverted after transformation into Escherichia coli. Interestingly, the inverted portion was found to be flanked by inverted repeats, and sealing of nicks inside the plasmid could correct the inversion. This type of sgRNA recombination completely changed its original sequence and should be noted during sgRNA design and performance of CRISPR/Cas9.

Keywords: CRISPR/Cas9; Inversion; Inverted repeats; sgRNA.

MeSH terms

Base Sequence
CRISPR-Cas Systems*
Escherichia coli / genetics*
Gene Editing* / methods
Gene Knockout Techniques / methods
Homeodomain Proteins / genetics
Humans
Inverted Repeat Sequences*
RNA, Guide, CRISPR-Cas Systems / genetics*
Transformation, Genetic

Substances

HOXB13 protein, human
Homeodomain Proteins
RNA, Guide, CRISPR-Cas Systems