Abstract
Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) that utilizes long single-stranded DNA (lssDNA) of 0.2–2 kbases in length as donor templates to insert large segments of novel DNA sequences or to replace endogenous genes at precise locations in the genome has enabled CRISPR-assisted genome editing to make strides toward a more simple and rapid workflow. By leveraging the notion that short single-stranded DNA oligo (<200 bases) serves as efficient donor in mouse zygotes for facilitating HDR-mediated genome editing, Easi-CRISPR expands to use lssDNA as the donor which accelerates the timeline to as little as 2 months for creating most types of genetically engineered mouse models (F0). Our lab (CGERC) has adopted Easi-CRISPR for multiple loci to generate mouse models over the past three plus years since its introduction. Here, we use two genes as examples to illustrate a step-by-step protocol for generating two commonly used models, including a knock-in (insertion of a reporter gene plus GOI) as well as a conditional knock-out model (via exon floxing). This protocol will focus more on molecular biology aspect, particularly we demonstrate two recently developed methods for lssDNA procuration: (1) PCR-based Takara Bio kit with modifications; (2) plasmid-retrieval-based CRISPR-CLIP (CRISPR-Clipped LssDNA via Incising Plasmid). Both methods are devised to retain sequence fidelity in lssDNA generated. In addition, CRISPR-CLIP directly retrieves lssDNA from DNA plasmid without using restriction enzymes through a PCR-free system hence carries virtually no restriction on sequence complexity, further mitigating limitations discussed in the original Easi-CRISPR protocol. We have alternated the use between both methods when suitable and successfully generated lssDNA templates via CRISPR-CLIP up to 3.5 kbases patched with multiple highly repetitive sequences, which is otherwise challenging to maneuver. Along with certain other modified workflow presented herein, Easi-CRISPR can be adapted to be more straightforward while applicable to generate mouse models in broader scope. (Certain figures and text passages presented in this chapter are reproduced from Shola et al. (The CRISPR J 3(2):109–122, 2020), published by Mary Ann Libert, Inc)
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Notes
- 1.
To maintain confidentiality of the research, partial sequences of the genes have been altered and some data shown herein are schematic for illustration purpose.
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Acknowledgments
We thank our lab (CGERC) members Vhy-Shelta Kewaldar, Pradip Kar, and Jing Gao for their help and assistance with the experiments; Transgenic (TRTC) members Jahnney Torres, Roxana Cubias, William Ramirez, and Eunyoung Kim for zygote microinjection and mouse husbandry; Patrick Smith and David Knorr (LMGI) for collaborative efforts in trying out the Easi-CRISPR system while we were at early stage establishing relevant methods and protocols. We gratefully acknowledge the support and help of Ravi Tolwani, Associate Vice President, Comparative Bioscience Center of the Rockefeller University, for providing resources to make this manuscript possible. We also give our thanks to Betty Shih, Enlightagen Consulting for the editorial contribution and helpful discussions on the manuscript.
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Shola, D.T.N., Yang, C., Han, C., Norinsky, R., Peraza, R.D. (2021). Generation of Mouse Model (KI and CKO) via Easi-CRISPR. In: Singh, S.R., Hoffman, R.M., Singh, A. (eds) Mouse Genetics . Methods in Molecular Biology, vol 2224. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1008-4_1
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DOI: https://doi.org/10.1007/978-1-0716-1008-4_1
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