https://www.alphaknockout.com Mouse Carmil2 Knockout Project (CRISPR/Cas9) Objective: To create a Carmil2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Carmil2 gene (NCBI Reference Sequence: NM_001357333.1 ; Ensembl: ENSMUSG00000050357 ) is located on Mouse chromosome 8. 39 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 39 (Transcript: ENSMUST00000213019). Exon 1~33 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: Mice homozygous for a knock-out or ENU-induced allele exhibit decreased regulatory T cells and reduced proliferative and IL2-secretion response to anti-CD3 and anti-CD28 antibodies. Exon 1 starts from about 0.02% of the coding region. Exon 1~33 covers 85.68% of the coding region. The size of effective KO region: ~9501 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' 10 12 14 16 18 25 27 29 1 2 3 4 5 6 7 8 9 11 13 15 17 24 26 28 30 31 32 33 11 11 39 Legends Exon of mouse Carmil2 Knockout region Exon of mouse Acd 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 start codon 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 2000 bp section downstream of Exon 33 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(25.05% 501) | C(25.35% 507) | T(24.15% 483) | G(25.45% 509) Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(23.3% 466) | C(25.25% 505) | T(24.65% 493) | G(26.8% 536) Note: The 2000 bp section downstream of Exon 33 is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. 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% chr8 + 105684275 105686274 2000 browser details YourSeq 147 1221 1411 2000 92.9% chr12 + 80625937 80626131 195 browser details YourSeq 146 1238 1412 2000 94.6% chr4 - 117180491 117181032 542 browser details YourSeq 145 1219 1396 2000 92.7% chr19 - 37399374 37399550 177 browser details YourSeq 141 1238 1448 2000 93.3% chr17 - 29987131 29987731 601 browser details YourSeq 139 1221 1397 2000 93.2% chr1 - 80436133 80436313 181 browser details YourSeq 139 1238 1777 2000 82.3% chr4 + 83415221 83415525 305 browser details YourSeq 136 1237 1396 2000 92.8% chr5 - 144278869 144279027 159 browser details YourSeq 136 1223 1396 2000 93.0% chr4 - 117207050 117207223 174 browser details YourSeq 136 1222 1396 2000 88.6% chr12 + 54736038 54736203 166 browser details YourSeq 135 1238 1396 2000 93.4% chr6 - 120182718 120182875 158 browser details YourSeq 135 1238 1396 2000 93.4% chr3 + 89466126 89466283 158 browser details YourSeq 135 1238 1396 2000 94.1% chr14 + 26055036 26055199 164 browser details YourSeq 134 1238 1397 2000 94.1% chr5 + 123165236 123165406 171 browser details YourSeq 134 1221 1396 2000 89.9% chr2 + 103619154 103619337 184 browser details YourSeq 134 1246 1397 2000 91.8% chr1 + 57102409 57102554 146 browser details YourSeq 133 1238 1396 2000 92.7% chr7 - 3439924 3440081 158 browser details YourSeq 133 1238 1396 2000 93.5% chr17 - 29008944 29009102 159 browser details YourSeq 133 1237 1397 2000 93.5% chr3 + 4741670 4741834 165 browser details YourSeq 133 1238 1396 2000 90.0% chr14 + 11292776 11292928 153 Note: The 2000 bp section upstream of start codon 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 2000 1 2000 2000 100.0% chr8 + 105695776 105697775 2000 browser details YourSeq 107 291 446 2000 87.0% chr14 + 54525655 54526210 556 browser details YourSeq 105 285 449 2000 87.4% chr1 - 153468187 153468365 179 browser details YourSeq 104 296 452 2000 85.1% chr10 - 26248159 26248330 172 browser details YourSeq 104 304 450 2000 89.0% chr13 + 55806680 55806838 159 browser details YourSeq 103 286 450 2000 87.7% chr5 + 34386491 34386912 422 browser details YourSeq 101 282 834 2000 92.5% chr9 - 66282006 66282630 625 browser details YourSeq 101 283 442 2000 85.6% chr9 + 27235643 27235825 183 browser details YourSeq 101 304 451 2000 89.9% chr7 + 29294848 29295001 154 browser details YourSeq 101 300 451 2000 90.4% chr1 + 157485808 157485960 153 browser details YourSeq 99 308 446 2000 88.4% chr6 - 30388134 30388283 150 browser details YourSeq 99 304 446 2000 87.4% chr3 - 95843272 95843425 154 browser details YourSeq 97 284 451 2000 85.6% chr3 + 104808459 104808631 173 browser details YourSeq 97 304 445 2000 86.0% chr16 + 22672494 22672646 153 browser details YourSeq 97 289 446 2000 85.3% chr14 + 103381823 103381992 170 browser details YourSeq 96 309 446 2000 91.4% chr3 - 153671819 153671968 150 browser details YourSeq 96 294 449 2000 88.3% chr3 - 123020255 123020426 172 browser details YourSeq 96 300 451 2000 85.1% chr17 - 74803496 74803660 165 browser details YourSeq 96 304 451 2000 85.2% chr7 + 24301401 24301568 168 browser details YourSeq 96 302 446 2000 86.1% chr7 + 24597747 24597906 160 Note: The 2000 bp section downstream of Exon 33 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Carmil2 capping protein regulator and myosin 1 linker 2 [ Mus musculus (house mouse) ] Gene ID: 234695, updated on 26-Jun-2020 Gene summary Official Symbol Carmil2 provided by MGI Official Full Name capping protein regulator and myosin 1 linker 2 provided by MGI Primary source MGI:MGI:2685431 See related Ensembl:ENSMUSG00000050357 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 Gm585; Rltpr; CG1399-PB; D130029J02Rik Expression Biased expression in thymus adult (RPKM 117.5), spleen adult (RPKM 27.1) and 3 other tissues See more Orthologs human all Genomic context Location: 8; 8 D3 See Carmil2 in Genome Data Viewer Exon count: 38 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (105686010..105698195) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (108214806..108222065) Chromosome 8 - NC_000074.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Carmil2 ENSMUSG00000050357 Description capping protein regulator and myosin 1 linker 2 [Source:MGI Symbol;Acc:MGI:2685431] Gene Synonyms D130029J02Rik, Rltpr Location Chromosome 8: 105,686,274-105,698,187 forward strand. GRCm38:CM001001.2 About this gene This gene has 3 transcripts (splice variants), 149 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Carmil2-203 ENSMUST00000213019.1 4194 1397aa ENSMUSP00000148422.1 Protein coding - S0DHL8 TSL:1 GENCODE basic APPRIS P1 Carmil2-201 ENSMUST00000062574.7 2526 No protein - Retained intron - - TSL:1 Carmil2-202 ENSMUST00000212643.1 2235 No protein - Retained intron - - TSL:5 Page 7 of 9 https://www.alphaknockout.com 31.91 kb Forward strand 105.68Mb 105.69Mb 105.70Mb Genes (Comprehensive set... Ctcf-204 >processed transcript Carmil2-203 >protein coding Pard6a-206 >processed transcript Ctcf-201 >protein coding Carmil2-201 >retained intron Pard6a-207 >retained intron Ctcf-206 >retained intron Carmil2-202 >retained intron Pard6a-202 >protein coding Ctcf-205 >processed transcript Pard6a-204 >protein coding Pard6a-201 >protein coding Pard6a-203 >protein coding Pard6a-205 >protein coding 4933405L10Rik-203 >processed transcript Contigs < AC152826.2 Genes < Acd-202nonsense mediated decay < Enkd1-204retained intron (Comprehensive set... < Acd-201protein coding < Enkd1-202nonsense mediated decay < Acd-205retained intron < Enkd1-203retained intron < Acd-204nonsense mediated decay < Acd-203nonsense mediated decay < Enkd1-201protein coding Regulatory Build 105.68Mb 105.69Mb 105.70Mb Reverse strand 31.91 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000213019 11.87 kb Forward strand Carmil2-203 >protein coding ENSMUSP00000148... MobiDB lite Low complexity (Seg) Superfamily SSF52047 SMART SM00368 Pfam CARMIL, pleckstrin homology domain CARMIL, C-terminal domain Leucine-rich repeat PANTHER PTHR24112 F-actin-uncapping protein RLTPR Gene3D Leucine-rich repeat domain superfamily PH-like domain superfamily All sequence SNPs/i..