Unpredicted central inversion in a sgRNA flanked by inverted repeats
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Unpredicted central inversion in a sgRNA flanked by inverted repeats Guannan Wang1,2,3 · Saraswati Sukumar1,4 Received: 23 October 2019 / Accepted: 14 May 2020 © Springer Nature B.V. 2020
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 · sgRNA · Inversion · Inverted repeats Abbreviations sgRNA Small guide RNA CRISPR Clustered regularly interspaced short palindromic repeat CRISPR/Cas9, the most widely used genome engineering approach at present, is employed in various biological applications, including gene knockout, activation or repression of target genes, and genome-wide screens, etc. The ~ 20 nucleotide sgRNA defines the genomic DNA to be modified [1],
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11033-020-05524-1) contains supplementary material, which is available to authorized users. * Guannan Wang [email protected] * Saraswati Sukumar [email protected] 1
Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
2
Department of Breast and Thyroid Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei, China
3
Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Rd NW, New Research Building, Room E512, Washington, D.C. 20007, USA
4
Department of Oncology, Johns Hopkins University School of Medicine, 1650 Orleans Street, CRB1/Room 143, Baltimore, MD 21287, USA
that no recombination of the sgRNA occurs must be guaranteed during transformation. When we designed the sgRNA to knock out human HOXB13 gene, we uploaded the DNA sequence from exon one to the ‘Guide Design Resources’, an online tool developed from Zhang lab, (https://zlab.bio/guide-design-resou rces). The rank No. 1 sgRNA the program suggested was CAAGGATATCGAAGGCTTGC. For the oligo design of this sgRNA, we added overhangs such that sticky ends are created after BsmBI (NEB, R0580S) digestion of the lentiCRISPR v2 plasmid (Addgene, #52,961) and a ‘G’ for U6 promoter transcription. The sequence of the oligos were 5′CACC GCA AGGATATCGAAG GCT TGC 3′ and 5′AAA C GC A AG C CT T CG ATAT CC T TG C 3′. The oligos were ordered from Thermo Fisher Scientific without modification. Oligos were annealed by slowly cooling from 95 °C to 25 °C at 5 °C/min. The digested plasmid was not dephosphorylated and the oligos were not phosphorylated. Annealed oligos were liga
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