Crispr cas mediated genome engineering

IRAKM knockout mice were generated by Dr. Richard Flavell (Yale School of Medicine, New Haven) as described^{51 (link)}. IRAKM flox/flox mice were generated by our lab as described^{52 (link)}. Adipocyte-specific deletion of IRAKM (IRAKM^AKO) was obtained by breeding IRAKM flox/flox mice with Adiponectin-Cre transgenic mice (Jackson Laboratory, 028020). IRAKM kinase-dead(K205A) knock-in mice were generated by CRISPR/Cas-mediated genome engineering (Cyagen Biosciences). Six- to eight-week-old male mice were maintained on a high-fat diet composed of 60% kcal derived from fat (TD06414, Envigo) for 12 weeks, or on high carbohydrate (62% Sucrose)/zero-fat diet (TD03314, Envigo) for 8 weeks while normal mice maintained on either standard rodent chow diet (2918 Teklad Global 18% Protein Rodent Diet, Envigo). Animals were housed in a specific pathogen-free facility at a temperature of 21 °C, relative humidity of 50–70%, and under a constant 12-h light/dark cycle and given free access to food and water. All procedures using animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Cleveland Clinic (protocol number: 2020–2316). Ethical compliance with IACUC protocols and institute standards was maintained.

The Pacs2 cKI in C57BL/6J mice was created using CRISPR/Cas-mediated genome engineering (Cyagen Biosciences). The Hipp11 locus is located within an intergenic region between the Eif4enif1 and Drg1 genes on mouse chromosome 11. The mouse Pacs2 gene (NCBI Reference Sequence: NM_001291444.1) is located on mouse chromosome 12. For the KI model, the ‘alphaMHC_long promoter-Kozak-Mouse Pacs2 CDS-rBG pA’ cassette was inserted into the Hipp11 locus (approximately 0.7 kb 5' of Eif4enif1 gene and 4.5 kb 3' of the Drg1 gene). To engineer the targeting vector, homology arms were generated by PCR using a BAC clone as the template. Cas9 and gRNA were co-injected into fertilized eggs with a targeting vector for mice production. The pups were genotyped by PCR followed by sequencing analysis.

The study protocol was approved by the Research Ethics Board of Sun Yat-sen memorial hospital of Sun Yat-sen University and Guangdong Provincial People’s Hospital. All the experimental procedures were approved by the Committee for Animal Research of Sun Yat-sen University and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
Four-week-old male C57BL/6 wild-type mice (n = 6) were purchased from the animal research center of Sun Yat-sen University. Whole-body LNK-deficient in C57BL/6 genetic backgrounds mice (n = 7) were created via CRISPR/Cas mediated genome engineering by Cyagen Biosciences Inc. The mouse Sh2b3 gene (GenBank accession number: NM_001306127.1; Ensembl: ENSMUSG00000042594) is located on mouse chromosome 5. Exon 1 to exon 3 were selected as target sites. Cas9 mRNA and gRNA generated using an in vitro transcription were then injected into fertilized eggs for knockout mouse production. All mice were randomly divided into different groups, housed 4 to 5 per cage, with standard laboratory conditions (12 h light:12 h darkness cycle) at a controlled temperature (23 ± 2 °C) and free access to rodent feed and water. All mice (4–5 weeks old) were fed a high-fat diet (HFD, 60% calories from fat, D12492; Research Diets Inc., New Brunswick, NJ, USA) for 16 weeks.

The Ttc6-knockout mouse model was generated on a C57BL/6 background using CRISPR/Cas-mediated genome engineering (Cyagen Biosciences Inc., Guangzhou, China). Based on the Ttc6 gene sequence (Ensembl: ENSMUSG00000046782), exon 2 to exon 32 was selected as target site and was deleted. For the production of knockout mice, Cas9 mRNA and gRNA, synthesized through in vitro transcription, were subsequently injected into fertilized eggs. The genotyping of the resultant mice was performed using PCR, followed by an examination through DNA sequencing analysis. In this study, RT-PCR experiments were also used to determine the Ttc6 genotype. All the genotyping primers were as follows: for PCR and DNA sequencing, forward 5′-CAG CTA CGG GGC ATG GAG GTA C-3′ and 5′-CTG AAA TCC TAC ACT TGT GAG ACT GGA GC-3′ and reverse 5′-AAC ATA TTC AAC AGT ATC CAG CTT GAA AAG A-3′; for RT-PCR, forward 5′-AAC TCG TGC CAT CCA TCT CC-3′ and reverse 5′-GGG ATT TGC TTT CAC GGC TT-3′. All procedures involving animal care and experiments were examined and sanctioned by the Animal Use Committee at the School of Medicine, Shandong University. Compliance with all relevant institutional and national guidelines for the humane treatment and utilization of animals was strict.