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Mouse Mis18bp1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Mis18bp1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Mis18bp1 (NCBI Reference Sequence: NM_172578 ; Ensembl: ENSMUSG00000047534 ) is located on Mouse 12. 14 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000052201). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Mis18bp1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-147P15 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 33.5% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 1329 bp, and the size of intron 5 for 3'-loxP site insertion: 825 bp. The size of effective cKO region: ~2847 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 3 4 5 6 7 14 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Mis18bp1 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(9347bp) | A(30.47% 2848) | C(17.52% 1638) | T(33.48% 3129) | G(18.53% 1732)

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% chr12 - 65157316 65160315 3000 browser details YourSeq 159 512 930 3000 81.6% chr15 - 85304158 85304491 334 browser details YourSeq 156 69 375 3000 84.0% chr8 - 85661380 85661780 401 browser details YourSeq 150 57 540 3000 86.4% chr15 - 99957980 99958425 446 browser details YourSeq 138 57 315 3000 86.3% chr11 + 101490645 101490897 253 browser details YourSeq 137 54 237 3000 87.5% chr2 - 157084029 157084214 186 browser details YourSeq 136 77 314 3000 86.6% chr15 - 38191258 38191682 425 browser details YourSeq 136 70 279 3000 90.4% chr9 + 115265354 115265912 559 browser details YourSeq 136 526 984 3000 78.0% chr15 + 95182067 95182486 420 browser details YourSeq 133 69 344 3000 85.1% chr7 - 80003065 80003460 396 browser details YourSeq 131 87 378 3000 90.8% chr2 + 155933315 155933852 538 browser details YourSeq 127 57 237 3000 85.1% chr1 - 192839435 192839614 180 browser details YourSeq 127 77 237 3000 90.0% chr1 - 88765721 88765883 163 browser details YourSeq 127 126 367 3000 90.6% chr4 + 129969216 129969491 276 browser details YourSeq 126 57 230 3000 88.0% chr19 - 37227180 37227356 177 browser details YourSeq 126 55 315 3000 90.5% chr4 + 33341935 33342423 489 browser details YourSeq 126 56 236 3000 84.2% chr18 + 35026437 35026616 180 browser details YourSeq 125 51 237 3000 83.5% chr7 - 83045703 83045883 181 browser details YourSeq 123 67 245 3000 81.3% chr4 + 41559955 41560125 171 browser details YourSeq 123 560 1111 3000 78.1% chr16 + 70749375 70749737 363

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% chr12 - 65151469 65154468 3000 browser details YourSeq 152 291 616 3000 94.3% chr3 - 27144045 27144463 419 browser details YourSeq 146 282 443 3000 93.2% chr1 - 60387443 60387602 160 browser details YourSeq 145 285 449 3000 94.5% chr11 - 98990739 98990904 166 browser details YourSeq 143 291 446 3000 96.1% chr17 - 31261869 31262031 163 browser details YourSeq 142 282 443 3000 94.4% chr3 + 120427445 120427612 168 browser details YourSeq 142 285 434 3000 96.0% chr3 + 104543177 104543325 149 browser details YourSeq 141 291 442 3000 96.7% chr17 - 51532832 51532983 152 browser details YourSeq 140 292 455 3000 89.9% chr2 - 130666811 130666967 157 browser details YourSeq 140 291 446 3000 97.4% chr11 - 51545455 51545611 157 browser details YourSeq 140 287 443 3000 94.9% chr10 - 70352000 70352156 157 browser details YourSeq 140 291 443 3000 96.1% chr6 + 29186618 29186771 154 browser details YourSeq 139 291 446 3000 94.9% chr5 - 127860544 127860701 158 browser details YourSeq 139 294 443 3000 96.7% chr1 - 134068855 134069016 162 browser details YourSeq 139 269 437 3000 94.3% chr15 + 35926143 35926727 585 browser details YourSeq 139 291 448 3000 91.7% chr10 + 88396408 88396562 155 browser details YourSeq 138 282 427 3000 95.9% chrX - 101631542 101631686 145 browser details YourSeq 138 285 443 3000 93.7% chr1 - 152402277 152402435 159 browser details YourSeq 138 291 440 3000 94.0% chr1 - 86194634 86194781 148 browser details YourSeq 138 272 431 3000 91.0% chr12 + 21453082 21453237 156

Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Mis18bp1 MIS18 binding protein 1 [ Mus musculus (house mouse) ] Gene ID: 217653, updated on 12-Aug-2019

Gene summary

Official Symbol Mis18bp1 provided by MGI Official Full Name MIS18 binding protein 1 provided by MGI Primary source MGI:MGI:2145099 See related Ensembl:ENSMUSG00000047534 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Knl2; C79407; mKIAA1903; 6720403H11 Expression Biased expression in CNS E11.5 (RPKM 12.0), liver E14 (RPKM 9.4) and 9 other tissues See more Orthologs human all

Genomic context

Location: 12; 12 C1 See Mis18bp1 in Genome Data Viewer

Exon count: 14

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (65132734..65172581, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (66233721..66273567, complement)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 8 transcripts

Gene: Mis18bp1 ENSMUSG00000047534

Description MIS18 binding protein 1 [Source:MGI Symbol;Acc:MGI:2145099] Gene Synonyms C79407 Location Chromosome 12: 65,132,734-65,172,604 reverse strand. GRCm38:CM001005.2 About this gene This gene has 8 transcripts (splice variants), 190 orthologues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Mis18bp1- ENSMUST00000052201.8 4017 998aa ENSMUSP00000052109.8 Protein coding CCDS25942 Q80WQ8 TSL:1 201 GENCODE basic APPRIS P1

Mis18bp1- ENSMUST00000222244.1 2053 434aa ENSMUSP00000152490.1 Protein coding - Q80WQ8 TSL:1 208 GENCODE basic

Mis18bp1- ENSMUST00000221296.1 969 168aa ENSMUSP00000152132.1 Protein coding - A0A1Y7VMX3 CDS 3' 207 incomplete TSL:5

Mis18bp1- ENSMUST00000124201.1 717 54aa ENSMUSP00000152203.1 Protein coding - A0A1Y7VN23 TSL:3 202 GENCODE basic

Mis18bp1- ENSMUST00000140391.7 5016 No - Retained - - TSL:5 204 protein intron

Mis18bp1- ENSMUST00000131753.1 2972 No - Retained - - TSL:1 203 protein intron

Mis18bp1- ENSMUST00000141456.1 601 No - Retained - - TSL:3 205 protein intron

Mis18bp1- ENSMUST00000149986.1 517 No - Retained - - TSL:2 206 protein intron

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59.87 kb Forward strand

65.13Mb 65.14Mb 65.15Mb 65.16Mb 65.17Mb 65.18Mb Fancm-207 >nonsense mediated decay 4930471E15Rik-201 >lncRNA (Comprehensive set...

Fancm-201 >protein coding

Fancm-202 >protein coding

Contigs < AC159621.2 < AC159885.2 Genes (Comprehensive set... < Mis18bp1-204retained intron

< Mis18bp1-201protein coding

< Mis18bp1-205retained intron < Mis18bp1-208protein coding

< Mis18bp1-207protein coding

< Mis18bp1-206retained intron

< Mis18bp1-202protein coding

< Mis18bp1-203retained intron

Regulatory Build

65.13Mb 65.14Mb 65.15Mb 65.16Mb 65.17Mb 65.18Mb Reverse strand 59.87 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000052201

< Mis18bp1-201protein coding

Reverse strand 39.85 kb

ENSMUSP00000052... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Homeobox-like domain superfamily

SMART SANT/Myb domain

Pfam SANT associated

PANTHER KNL2-like

PTHR16124:SF3 Gene3D 1.10.10.60 CDD SANT/Myb domain

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

Variant Legend

inframe deletion missense variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 998

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

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