https://www.alphaknockout.com Mouse Fcrl1 Knockout Project (CRISPR/Cas9) Objective: To create a Fcrl1 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 2~10 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 2 starts from about 2.94% of the coding region. Exon 2~10 covers 90.27% of the coding region. The size of effective KO region: ~8937 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 8 9 10 11 Legends Exon of mouse Fcrl1 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 952 bp section downstream of Exon 10 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(20.0% 400) | C(21.75% 435) | T(38.05% 761) | G(20.2% 404) Note: The 2000 bp section upstream of Exon 2 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(952bp) | A(27.63% 263) | C(22.48% 214) | T(29.31% 279) | G(20.59% 196) Note: The 952 bp section downstream of Exon 10 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 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr3 + 87379326 87381325 2000 browser details YourSeq 1075 404 1892 2000 88.5% chr8 - 104317988 104319468 1481 browser details YourSeq 1061 412 1868 2000 88.3% chr7 - 39660587 39662046 1460 browser details YourSeq 1059 412 1868 2000 88.0% chr6 - 119233964 119235405 1442 browser details YourSeq 1054 412 1892 2000 88.3% chr14 + 66372108 66373581 1474 browser details YourSeq 1051 412 1892 2000 87.7% chr19 + 38012835 38014285 1451 browser details YourSeq 1049 412 1868 2000 88.3% chr19 - 25029787 25031218 1432 browser details YourSeq 1045 418 1890 2000 87.6% chr15 - 10967973 10969419 1447 browser details YourSeq 1041 412 1868 2000 88.0% chr2 + 133028056 133029500 1445 browser details YourSeq 1040 413 1868 2000 89.6% chr18 + 57070258 57071775 1518 browser details YourSeq 1035 411 1835 2000 87.4% chr3 - 154404292 154405675 1384 browser details YourSeq 1030 412 1825 2000 87.9% chrX - 21043483 21411016 367534 browser details YourSeq 1030 445 1892 2000 87.9% chr7 - 3139539 3140964 1426 browser details YourSeq 1030 412 1890 2000 87.7% chr6 + 7148211 7149658 1448 browser details YourSeq 1027 417 1856 2000 87.9% chr8 - 79237760 79239190 1431 browser details YourSeq 1027 412 1859 2000 88.7% chr5 + 130415917 130417337 1421 browser details YourSeq 1026 411 1857 2000 87.3% chr7 + 83507202 83508627 1426 browser details YourSeq 1026 412 1858 2000 88.5% chr5 + 77936145 77937574 1430 browser details YourSeq 1026 411 1859 2000 87.1% chr17 + 56526838 56528256 1419 browser details YourSeq 1024 412 1868 2000 87.9% chr18 - 66891108 66892536 1429 Note: The 2000 bp section upstream of Exon 2 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 952 1 952 952 100.0% chr3 + 87390263 87391214 952 browser details YourSeq 62 423 589 952 84.6% chr9 + 94616476 94616641 166 browser details YourSeq 60 502 589 952 88.7% chr17 + 84175403 84175740 338 browser details YourSeq 55 484 583 952 91.4% chr1 + 118970214 118970314 101 browser details YourSeq 54 420 511 952 80.7% chr1 - 166265141 166265233 93 browser details YourSeq 51 478 591 952 79.2% chrX - 12896675 12896781 107 browser details YourSeq 51 464 616 952 76.3% chr13 + 13578631 13578752 122 browser details YourSeq 50 464 589 952 88.1% chr5 + 135748991 135749119 129 browser details YourSeq 48 409 589 952 76.4% chr1 - 58013145 58013322 178 browser details YourSeq 46 485 567 952 83.4% chr7 - 131014226 131014306 81 browser details YourSeq 46 409 495 952 82.9% chr12 + 55525535 55525622 88 browser details YourSeq 45 480 585 952 71.5% chr1 + 128023016 128023137 122 browser details YourSeq 44 425 524 952 72.3% chr12 - 102134441 102134539 99 browser details YourSeq 44 425 495 952 83.4% chr1 + 180621188 180621257 70 browser details YourSeq 43 409 495 952 95.9% chr2 - 93129619 93129707 89 browser details YourSeq 42 505 572 952 80.9% chr2 + 118576810 118576877 68 browser details YourSeq 41 478 580 952 93.7% chr4 + 98846691 98846805 115 browser details YourSeq 41 437 497 952 83.7% chr4 + 58519138 58519198 61 browser details YourSeq 41 448 567 952 97.7% chr1 + 150621885 150622004 120 browser details YourSeq 40 505 565 952 83.7% chr13 + 14219425 14219486 62 Note: The 952 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 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 6 of 9 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 7 of 9 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 8 of 9 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 9 of 9.