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Mouse Tcaim Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tcaim conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tcaim gene (NCBI Reference Sequence: NM_001013405 ; Ensembl: ENSMUSG00000046603 ) is located on Mouse chromosome 9. 11 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 11 (Transcript: ENSMUST00000052740). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tcaim gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-119I16 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 3 starts from about 3.93% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 2921 bp, and the size of intron 3 for 3'-loxP site insertion: 2538 bp. The size of effective cKO region: ~636 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 11 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tcaim Homology arm cKO region loxP site

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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(7136bp) | A(29.69% 2119) | C(19.65% 1402) | T(29.86% 2131) | G(20.8% 1484)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr9 + 122808509 122811508 3000 browser details YourSeq 247 1077 1606 3000 82.4% chr4 - 53268942 53269581 640 browser details YourSeq 224 1072 1616 3000 85.4% chr6 - 54349936 54350516 581 browser details YourSeq 220 1092 1704 3000 82.8% chr2 + 130155106 130155959 854 browser details YourSeq 212 1077 1618 3000 83.4% chr3 - 100391758 100392350 593 browser details YourSeq 203 1081 1463 3000 86.6% chr2 - 83898665 83899058 394 browser details YourSeq 195 1092 1531 3000 81.5% chr2 + 74247603 74248059 457 browser details YourSeq 193 1076 1590 3000 79.9% chr14 + 104788439 104789168 730 browser details YourSeq 189 1080 1557 3000 83.7% chr19 - 24710524 24800105 89582 browser details YourSeq 187 1078 1539 3000 83.3% chr2 - 38455907 38456350 444 browser details YourSeq 186 1079 1536 3000 85.5% chr1 - 166979262 166979751 490 browser details YourSeq 184 1077 1611 3000 84.8% chr12 - 38983455 38984003 549 browser details YourSeq 183 1078 1561 3000 83.3% chr17 - 42964287 42965202 916 browser details YourSeq 183 1078 1579 3000 89.3% chr12 + 58260265 58260778 514 browser details YourSeq 178 1084 1622 3000 79.3% chr7 - 118102594 118103244 651 browser details YourSeq 176 1092 1536 3000 81.6% chr10 - 8018068 8018822 755 browser details YourSeq 172 1088 1585 3000 84.4% chrX - 135242092 135242854 763 browser details YourSeq 172 1077 1602 3000 91.0% chr9 - 93947599 93948205 607 browser details YourSeq 171 1063 1464 3000 83.3% chr2 + 72755187 72755623 437 browser details YourSeq 170 1130 1691 3000 88.0% chr4 - 53988651 54261515 272865

Note: The 3000 bp section upstream of Exon 3 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% chr9 + 122812145 122815144 3000 browser details YourSeq 76 1950 2165 3000 92.3% chr7 - 30327044 30492879 165836 browser details YourSeq 64 1907 2022 3000 80.4% chr17 + 84713405 84713546 142 browser details YourSeq 62 1092 1237 3000 87.0% chr1 - 179156044 179156355 312 browser details YourSeq 61 1108 1198 3000 83.6% chr3 + 117292665 117292755 91 browser details YourSeq 60 1907 2007 3000 84.9% chr10 - 40337828 40337939 112 browser details YourSeq 55 1891 2013 3000 88.6% chr9 - 74044318 74044636 319 browser details YourSeq 53 1907 1991 3000 84.5% chr7 - 142030484 142030569 86 browser details YourSeq 52 1909 1996 3000 82.5% chr4 - 33678384 33678472 89 browser details YourSeq 48 1907 1993 3000 84.3% chr4 + 109047170 109047256 87 browser details YourSeq 47 1907 1993 3000 79.8% chr4 - 124697136 124697223 88 browser details YourSeq 45 1879 1986 3000 68.2% chr1 - 125588276 125588360 85 browser details YourSeq 45 1107 1240 3000 75.6% chrX + 12546445 12546552 108 browser details YourSeq 44 1875 1970 3000 73.0% chr1 - 185843353 185843443 91 browser details YourSeq 44 1932 2013 3000 78.6% chr1 + 189039975 189040050 76 browser details YourSeq 44 1932 2019 3000 88.0% chr1 + 93776277 93776798 522 browser details YourSeq 42 900 1186 3000 95.7% chr4 - 3615670 3616208 539 browser details YourSeq 42 1953 2133 3000 92.0% chr11 - 106958574 106958951 378 browser details YourSeq 42 1907 1991 3000 77.2% chr1 - 16516811 16516894 84 browser details YourSeq 42 1906 1968 3000 88.9% chr12 + 35926097 35926161 65

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tcaim T cell activation inhibitor, mitochondrial [ Mus musculus (house mouse) ] Gene ID: 382117, updated on 12-Aug-2019

Gene summary

Official Symbol Tcaim provided by MGI Official Full Name T cell activation inhibitor, mitochondrial provided by MGI Primary source MGI:MGI:1196217 See related Ensembl:ENSMUSG00000046603 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 Gm1129; TOAG-1; D9Ertd402e Expression Ubiquitous expression in heart adult (RPKM 4.1), kidney adult (RPKM 3.1) and 26 other tissues See more Orthologs human all

Genomic context

Location: 9 F4; 9 73.14 cM See Tcaim in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (122805513..122838924)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (122714665..122745450)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 4 transcripts

Gene: Tcaim ENSMUSG00000046603

Description T cell activation inhibitor, mitochondrial [Source:MGI Symbol;Acc:MGI:1196217] Gene Synonyms D9Ertd402e, LOC382117 Location Chromosome 9: 122,805,539-122,836,334 forward strand. GRCm38:CM001002.2 About this gene This gene has 4 transcripts (splice variants), 203 orthologues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tcaim- ENSMUST00000052740.13 3049 509aa ENSMUSP00000049759.7 Protein coding CCDS23647 G3X983 TSL:1 201 GENCODE basic APPRIS P1

Tcaim- ENSMUST00000136274.2 2909 126aa ENSMUSP00000120948.2 Nonsense mediated - D6RHM0 TSL:5 202 decay

Tcaim- ENSMUST00000213178.1 1580 No - Retained intron - - TSL:NA 204 protein

Tcaim- ENSMUST00000149659.1 377 No - lncRNA - - TSL:3 203 protein

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50.80 kb Forward strand 122.80Mb 122.81Mb 122.82Mb 122.83Mb 122.84Mb Genes (Comprehensive set... Topaz1-201 >protein coding Tcaim-201 >protein coding

Tcaim-203 >lncRNA Gm25341-201 >misc RNA

Tcaim-202 >nonsense mediated decay

Tcaim-204 >retained intron

Contigs < AC125374.4 Genes < Gm35549-204lncRNA (Comprehensive set...

< Gm35549-201protein coding

< Gm35549-202protein coding

< Gm35549-203protein coding

< Zfp445-205protein coding

Regulatory Build

122.80Mb 122.81Mb 122.82Mb 122.83Mb 122.84Mb Reverse strand 50.80 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000052740

30.80 kb Forward strand

Tcaim-201 >protein coding

ENSMUSP00000049... Pfam Domain of unknown function DUF4460 Domain of unknown function DUF4461

PANTHER PTHR31596:SF1

T-cell activation inhibitor, mitochondrial

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 509

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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