Crispr rna crrna

ApoE knockout mice were generated in zygotes from homozygous Rag2^tm1.1FlvCsf1^tm1(CSF1)FlvIl2rg^tm1.1FlvApp^tm3.1Tcs mice using CRISPR–Cas9 technology by targeting exon 4 of the mouse ApoE gene. The RNA guide 5′-CCTCGTTGCGGTACTGCCCGAGT-3′ was selected using the CRISPOR web tool. Ribonucleoproteins containing 0.3 μM purified Cas9HiFi protein (Integrated DNA Technologies), 0.3 μM CRISPR RNA (crRNA) and 0.3 μM trans-activating crRNA (Integrated DNA Technologies) were injected into the pronucleus of 72 embryos by microinjection in the Mouse Expertise Unit of KU Leuven. Two candidate pups were identified by PCR analysis with several primer combinations. One founder was selected for breeding and an allele with a chromosomal deletion of 335 base pairs (bp) (corresponding to 148 bp of intronic sequence and the first 187 bp of exon 4 sequence) (Extended Data Fig. 4) was selected to establish the colony. The founder mouse was backcrossed over two generations before a homozygous colony was established, which was designated App^NL-G-FApoE^−/−. The strain was maintained on the original C57Bl6J:BalbC background. Standard genotyping for the ApoE allele was performed by PCR with primers 5′-GCTCCCAAGTCACACAAGAA-3′ and 5′-CTCACGGATGGCACTCAC-3′, resulting in a 755-bp amplicon for the WT allele and a 420-bp amplicon for the ApoE knockout allele.