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A cost efficient protocol to introduce epitope tags by CRISPR-Cas9 mediated gene knock-in with asymmetric semi-double stranded template.

ABSTRACT: To study the biological function of uncharacterized proteins, specific antibodies are in high demand. However, the production of desirable antibodies such as highly specific or high affinity is not always successful. Furthermore, even if commercially available antibodies exist, the cost, quality, and accessibility often differ from country to country. In comparison, epitope tags are reliable and economical options since good antibodies against major epitope tags are commercially available. Although exogenously expressed epitope-tagged protein appears as a timely method, the excessive protein production may not faithfully recapitulate its biology. Thanks to the recent advances in genome editing by CRISPR-Cas9, HDR-mediated endogenous protein tagging has become an accessible approach for many labs. However, currently the synthesis of long (>100 bp), chemically modified oligos can be time-consuming and costly. To develop a reliable, simple, and cost-effective epitope-tagging method that requires minimal materials and apparatus, we focus on an approach utilizing two non-chemically modified shorter-annealed oligos (semi-dsODNs) mediated HDR for epitope tags insertion. We also use a cationic lipid chemical, polyethyleneimine (PEI), for plasmid delivery to minimize the cost and materials used while a considerable success rate could be achieved. • This protocol provides a more economical way to generate CRISPR-Cas9 mediated gene knock-in. • This protocol provides a simplified design of semi-dsODN without chemical modification on the oligos. • This protocol provides a simplified experimental procedure. In vitro assembled Cas9 complex and electroporation are not required.

SUBMITTER: Fang Q

PROVIDER: S-EPMC8374323 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an ∼200 base-pair sequence encoding a composite tag can be efficiently "knocked-in" into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the sox3, sox11a, and pax6a genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3' homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the sox3 and pax6a knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited sox3, sox11a, and pax6a knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5' and 3' junctions indicated that knock-in allele frequencies were higher at the 3' side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos.

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A cost efficient protocol to introduce epitope tags by CRISPR-Cas9 mediated gene knock-in with asymmetric semi-double stranded template.

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