https://www.alphaknockout.com Mouse Fcrl1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Fcrl1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fcrl1 gene (NCBI Reference Sequence: NM_153090 ; Ensembl: ENSMUSG00000059994 ) is located on Mouse chromosome 3. 11 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 11 (Transcript: ENSMUST00000163661). Exon 4~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Fcrl1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-61O7 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 4 starts from about 10.38% of the coding region. The knockout of Exon 4~7 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2806 bp, and the size of intron 7 for 3'-loxP site insertion: 2569 bp. The size of effective cKO region: ~2683 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 4 5 6 7 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Fcrl1 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(9183bp) | A(26.23% 2409) | C(23.75% 2181) | T(27.66% 2540) | G(22.36% 2053) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr3 + 87381339 87384338 3000 browser details YourSeq 143 1115 1326 3000 87.9% chr4 + 136037289 136037648 360 browser details YourSeq 136 1111 1328 3000 85.4% chr11 + 76453129 76453517 389 browser details YourSeq 134 1122 1304 3000 87.3% chr17 - 80087690 80087870 181 browser details YourSeq 134 1120 1328 3000 87.3% chr16 - 18439066 18439439 374 browser details YourSeq 130 1115 1304 3000 83.9% chrX - 151761289 151761472 184 browser details YourSeq 130 1120 1304 3000 85.0% chr1 - 157504147 157504324 178 browser details YourSeq 129 1120 1305 3000 85.5% chr5 - 122840691 122840870 180 browser details YourSeq 129 1115 1284 3000 90.1% chr4 - 155259306 155259477 172 browser details YourSeq 129 1115 1325 3000 89.1% chr1 - 37034166 37034793 628 browser details YourSeq 129 1120 1328 3000 82.6% chr11 + 73061080 73061261 182 browser details YourSeq 127 1120 1320 3000 81.7% chr12 + 3508458 3508637 180 browser details YourSeq 125 1112 1304 3000 83.7% chr13 + 40861693 40861880 188 browser details YourSeq 124 1142 1326 3000 88.5% chr11 - 115580218 115706231 126014 browser details YourSeq 124 1120 1293 3000 87.9% chr4 + 134260534 134260719 186 browser details YourSeq 123 1115 1304 3000 83.7% chr10 - 69257435 69257621 187 browser details YourSeq 123 1115 1304 3000 85.6% chr10 + 128570734 128823375 252642 browser details YourSeq 122 1115 1302 3000 85.1% chr9 - 66265158 66265342 185 browser details YourSeq 122 1153 1324 3000 89.7% chr16 - 31780496 31780866 371 browser details YourSeq 122 1120 1304 3000 83.0% chr1 + 40579011 40579187 177 Note: The 3000 bp section upstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr3 + 87387022 87390021 3000 browser details YourSeq 178 180 1648 3000 90.5% chr13 + 94934814 95337936 403123 browser details YourSeq 160 168 1470 3000 87.8% chr5 + 68022856 68185063 162208 browser details YourSeq 151 15 479 3000 75.9% chr5 - 9013736 9014207 472 browser details YourSeq 145 178 682 3000 88.1% chr6 - 84905866 84906492 627 browser details YourSeq 141 180 471 3000 79.1% chr6 - 87462206 87462487 282 browser details YourSeq 128 181 479 3000 85.9% chr7 - 134129395 134129680 286 browser details YourSeq 126 90 462 3000 82.4% chr19 + 58123498 58123866 369 browser details YourSeq 122 9 462 3000 90.8% chrX + 6350435 6350904 470 browser details YourSeq 116 80 451 3000 76.5% chr4 + 9127966 9128316 351 browser details YourSeq 113 239 461 3000 82.6% chr16 + 23352911 23353121 211 browser details YourSeq 111 172 611 3000 87.2% chr11 - 84477412 84477935 524 browser details YourSeq 107 237 479 3000 87.5% chr19 - 38083463 38083715 253 browser details YourSeq 106 15 475 3000 92.8% chr13 - 91110692 91111294 603 browser details YourSeq 106 301 478 3000 81.2% chr10 - 49761489 49761668 180 browser details YourSeq 106 15 523 3000 83.2% chr17 + 30126819 30127265 447 browser details YourSeq 106 185 512 3000 85.4% chr1 + 127886545 127886876 332 browser details YourSeq 105 9 479 3000 82.1% chrX - 92558295 92558741 447 browser details YourSeq 103 237 474 3000 81.6% chr5 - 126746816 126747012 197 browser details YourSeq 101 237 439 3000 80.0% chr5 - 105964701 105964892 192 Note: The 3000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Fcrl1 Fc receptor-like 1 [ Mus musculus (house mouse) ] Gene ID: 229499, updated on 12-Aug-2019 Gene summary Official Symbol Fcrl1 provided by MGI Official Full Name Fc receptor-like 1 provided by MGI Primary source MGI:MGI:2442862 See related Ensembl:ENSMUSG00000059994 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Bxmh1; Fcrh1; Ifgp1; Bxmas1; Bxmh1b; Fcrh1l; Fcrh1s; A230020G22Rik Expression Biased expression in spleen adult (RPKM 18.8), mammary gland adult (RPKM 4.3) and 1 other tissue See more Genomic context Location: 3; 3 F1 See Fcrl1 in Genome Data Viewer Exon count: 15 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (87376353..87403967) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (87180309..87196055) Chromosome 3 - NC_000069.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Fcrl1 ENSMUSG00000059994 Description Fc receptor-like 1 [Source:MGI Symbol;Acc:MGI:2442862] Gene Synonyms A230020G22Rik, BXMAS1-like, Fcrh1, IFGP1, mBXMH1, mIFGP1, moFcRH1, moFcRH1L, moFcRH1S Location Chromosome 3: 87,376,387-87,402,934 forward strand. GRCm38:CM000996.2 About this gene This gene has 6 transcripts (splice variants), 76 orthologues, 13 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Fcrl1-201 ENSMUST00000072480.8 1980 343aa ENSMUSP00000072300.2 Protein coding CCDS17452 A0A0R4J0W8 TSL:1 GENCODE basic APPRIS P3 Fcrl1-203 ENSMUST00000167200.6 1793 300aa ENSMUSP00000128235.1 Protein coding CCDS50942 E9Q5S9 TSL:1 GENCODE basic APPRIS ALT2 Fcrl1-202 ENSMUST00000163661.7 1649 363aa ENSMUSP00000130936.1 Protein coding CCDS50941 E9Q958 TSL:1 GENCODE basic APPRIS ALT2 Fcrl1-206 ENSMUST00000194786.5 1540 324aa ENSMUSP00000142286.1 Protein coding CCDS79937 A0A0A6YY56 TSL:1 GENCODE basic APPRIS ALT2 Fcrl1-204 ENSMUST00000191666.1 925 208aa ENSMUSP00000141916.1 Protein coding - A0A0A6YXB2 TSL:1 GENCODE basic APPRIS ALT2 Fcrl1-205 ENSMUST00000193854.1 2183 No protein - Retained intron - - TSL:2 Page 6 of 8 https://www.alphaknockout.com 46.55 kb Forward strand 87.37Mb 87.38Mb 87.39Mb 87.40Mb 87.41Mb Genes (Comprehensive set... Cd5l-201 >protein coding Fcrl1-202 >protein coding Fcrl1-201 >protein coding Fcrl1-203 >protein coding Fcrl1-206 >protein coding Fcrl1-204 >protein coding Fcrl1-205 >retained intron Contigs AC091682.26 > AC163618.4 > Regulatory Build 87.37Mb 87.38Mb 87.39Mb 87.40Mb 87.41Mb Reverse strand 46.55 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000163661 15.35 kb Forward strand Fcrl1-202 >protein coding ENSMUSP00000130... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily SMART Immunoglobulin subtype Pfam Immunoglobulin-like domain PROSITE profiles Immunoglobulin-like domain PANTHER PTHR11481:SF92 PTHR11481 Gene3D Immunoglobulin-like fold CDD cd00096 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 363 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.