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Mouse Bcl10 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Bcl10 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Bcl10 gene (NCBI Reference Sequence: NM_009740 ; Ensembl: ENSMUSG00000028191 ) is located on Mouse chromosome 3. 3 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000029842). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Bcl10 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-251M10 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: About one-third of homozygous null embryos die exhibiting exencephaly. Surviving mutants display immunological defects including severe immunodeficiency, abnormal B cell development and function, and impaired humoral response to bacterial infection.

Exon 2 starts from about 8.3% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 5890 bp, and the size of intron 2 for 3'-loxP site insertion: 2256 bp. The size of effective cKO region: ~789 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 2 3 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Bcl10 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7289bp) | A(26.45% 1928) | C(21.53% 1569) | T(28.51% 2078) | G(23.51% 1714)

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% chr3 + 145927157 145930156 3000 browser details YourSeq 125 1973 2109 3000 96.4% chr12 - 65865713 65865881 169 browser details YourSeq 120 1969 2101 3000 95.5% chr18 - 42069006 42069146 141 browser details YourSeq 118 1975 2108 3000 94.1% chr8 - 52378834 52378967 134 browser details YourSeq 117 1973 2100 3000 96.1% chr4 - 132528659 132528787 129 browser details YourSeq 117 1977 2107 3000 94.7% chr12 - 80485590 80485720 131 browser details YourSeq 116 1977 2104 3000 95.4% chr2 - 29770504 29770631 128 browser details YourSeq 115 1980 2107 3000 95.4% chr7 + 138370137 138370281 145 browser details YourSeq 113 1979 2107 3000 93.8% chr2 + 139987863 139987991 129 browser details YourSeq 112 1973 2109 3000 88.1% chr6 + 141220320 141220445 126 browser details YourSeq 108 1976 2109 3000 90.3% chr1 - 132181538 132181671 134 browser details YourSeq 105 1973 2103 3000 90.1% chr10 + 31419603 31419733 131 browser details YourSeq 71 1294 1473 3000 80.9% chr4 + 131337190 131337743 554 browser details YourSeq 70 1356 1473 3000 79.7% chr18 + 9693140 9693257 118 browser details YourSeq 64 1400 1507 3000 79.7% chr6 - 119854840 119854947 108 browser details YourSeq 60 1302 1438 3000 82.7% chr7 - 111878490 111878667 178 browser details YourSeq 60 1349 1479 3000 82.7% chr5 + 24028081 24028224 144 browser details YourSeq 59 1328 1437 3000 73.4% chr3 - 104360934 104361039 106 browser details YourSeq 58 1419 1506 3000 84.1% chr11 - 5138233 5138496 264 browser details YourSeq 58 1371 1479 3000 80.3% chr10 + 121520333 121520443 111

Note: The 3000 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 3000 1 3000 3000 100.0% chr3 + 145930946 145933945 3000 browser details YourSeq 891 2004 3000 3000 95.0% chr3 + 19585188 19586183 996 browser details YourSeq 89 1173 1616 3000 81.3% chr9 - 70733716 70734117 402 browser details YourSeq 64 1254 1564 3000 94.4% chr7 + 25261046 25263000 1955 browser details YourSeq 59 1440 1610 3000 90.5% chr11 - 34816152 34816341 190 browser details YourSeq 58 1149 1214 3000 97.0% chr10 - 127017100 127017174 75 browser details YourSeq 58 1143 1208 3000 90.8% chr10 - 100401231 100401295 65 browser details YourSeq 57 1473 1634 3000 88.0% chr14 + 73514165 73514385 221 browser details YourSeq 56 1173 1279 3000 95.2% chr7 + 80007607 80008056 450 browser details YourSeq 54 1441 1618 3000 93.6% chr2 - 18929372 18929562 191 browser details YourSeq 51 1146 1207 3000 94.9% chr18 - 28478212 28478282 71 browser details YourSeq 51 1158 1212 3000 92.6% chr10 + 26570883 26570936 54 browser details YourSeq 47 1461 1621 3000 96.3% chr1 - 54936665 54936836 172 browser details YourSeq 46 1523 1638 3000 81.9% chr14 + 50775976 50776087 112 browser details YourSeq 43 1421 1479 3000 93.9% chr8 + 124209183 124209243 61 browser details YourSeq 43 1445 1534 3000 82.1% chr10 + 127236384 127236482 99 browser details YourSeq 41 1825 1880 3000 88.7% chr10 + 61381155 61381226 72 browser details YourSeq 40 1460 1534 3000 97.7% chr4 - 86264875 86264969 95 browser details YourSeq 40 1460 1609 3000 95.5% chr1 - 134394711 134394860 150 browser details YourSeq 39 1441 1497 3000 84.5% chr6 - 28846829 28846882 54

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Bcl10 B cell leukemia/lymphoma 10 [ Mus musculus (house mouse) ] Gene ID: 12042, updated on 15-Oct-2019

Gene summary

Official Symbol Bcl10 provided by MGI Official Full Name B cell leukemia/lymphoma 10 provided by MGI Primary source MGI:MGI:1337994 See related Ensembl:ENSMUSG00000028191 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 CLAP; ME10; cE10; CIPER; BCL-10; C81403; CARMEN; AI132454 Expression Ubiquitous expression in large intestine adult (RPKM 10.2), bladder adult (RPKM 8.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 H2 See Bcl10 in Genome Data Viewer

Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (145924262..145934366)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (145587342..145597247)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 3 transcripts

Gene: Bcl10 ENSMUSG00000028191

Description B cell leukemia/lymphoma 10 [Source:MGI Symbol;Acc:MGI:1337994] Gene Synonyms BCL-10, cE10, mE10 Location Chromosome 3: 145,922,804-145,934,356 forward strand. GRCm38:CM000996.2 About this gene This gene has 3 transcripts (splice variants), 98 orthologues, is a member of 1 Ensembl protein family and is associated with 36 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Bcl10-201 ENSMUST00000029842.8 1949 233aa ENSMUSP00000029842.7 Protein coding CCDS17897 B7ZWE5 Q9Z0H7 TSL:1 GENCODE basic APPRIS P1

Bcl10-202 ENSMUST00000197842.1 2368 No protein - Retained intron - - TSL:NA

Bcl10-203 ENSMUST00000198122.1 660 No protein - lncRNA - - TSL:3

31.55 kb Forward strand 145.92Mb 145.93Mb 145.94Mb Genes (Comprehensive set... Bcl10-202 >retained intron 2410004B18Rik-202 >nonsense mediated decay

Bcl10-201 >protein coding 2410004B18Rik-204 >nonsense mediated decay

Bcl10-203 >lncRNA 2410004B18Rik-201 >protein coding

2410004B18Rik-206 >retained intron

2410004B18Rik-207 >protein coding

2410004B18Rik-203 >retained intron

2410004B18Rik-205 >protein coding

Contigs < AC123684.8 Regulatory Build

145.92Mb 145.93Mb 145.94Mb Reverse strand 31.55 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000029842

10.10 kb Forward strand

Bcl10-201 >protein coding

ENSMUSP00000029... MobiDB lite Low complexity (Seg) Superfamily Death-like domain superfamily Pfam CARD domain PROSITE profiles CARD domain PANTHER B-cell lymphoma/leukemia 10/E10 Gene3D 1.10.533.10 CDD BCL10, CARD domain

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 200 233

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

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