Generation of Mouse Model (KI and CKO) via Easi-CRISPR.

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).

Limitation of adipose tissue by the number of embryonic progenitor cells.

Adipogenesis in adulthood replaces fat cells that turn over and can contribute to the development of obesity. However, the proliferative potential of adipocyte progenitors in vivo is unknown (Faust et al., 1976; Faust et al., 1977; Hirsch and Han, 1969; Johnson and Hirsch, 1972). We addressed this by injecting labeled wild-type embryonic stem cells into blastocysts derived from lipodystrophic A-ZIP transgenic mice, which have a genetic block in adipogenesis. In the resulting chimeric animals, wild-type ES cells are the only source of mature adipocytes. We found that when chimeric animals were fed a high-fat-diet, animals with low levels of chimerism showed a significantly lower adipose tissue mass than animals with high levels of chimerism. The difference in adipose tissue mass was attributed to variability in the amount of subcutaneous adipose tissue as the amount of visceral fat was independent of the level of chimerism. Our findings thus suggest that proliferative potential of adipocyte precursors is limited and can restrain the development of obesity.