CLICK:One-step generation of conditional knockout mice

Abstract Background CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive. Results Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats. Conclusions The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice

Additional file 18: of CLICK: one-step generation of conditional knockout mice

Table S4. Primer sets used for PCR and sequencing analysis in this study. (XLSX 10 kb

Additional file 18: of CLICK: one-step generation of conditional knockout mice

Table S4. Primer sets used for PCR and sequencing analysis in this study. (XLSX 10 kb

Additional file 1: of CLICK: one-step generation of conditional knockout mice

Figure S1. Schematic representation of the protocol to prepare long single-stranded DNA (lssDNA) using nicking endonucleases. (AI 1147 kb

Additional file 5: of CLICK: one-step generation of conditional knockout mice

Figure S5. Floxed Tyr alleles generated by the microinjection of two gRNA, Cas9 mRNA and lssDNA(−) into B6 mouse zygotes. (AI 1656 kb

Additional file 9: of CLICK: one-step generation of conditional knockout mice

Figure S9. Sequences of two gRNAs and lssDNA(+) designed to target exon 3 of the mouse Mct4 locus. (AI 1209 kb

Additional file 17: of CLICK: one-step generation of conditional knockout mice

Table S3. Crossing floxed founders with Cre-driver mice. (XLSX 11 kb

Additional file 6: of CLICK: one-step generation of conditional knockout mice

Figure S6. Sequences of two gRNAs and lssDNA(−) designed to target exon 3 of the mouse KIAA1322 locus. (AI 1214 kb

Additional file 15: of CLICK: one-step generation of conditional knockout mice

Table S1. Germline transmission of CLICK-mediated mutations in F1 offspring. (XLSX 12 kb