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Mouse Sptb Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Sptb (NCBI Reference Sequence: NM_013675 ; Ensembl: ENSMUSG00000021061 ) is located on Mouse 12. 36 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 36 (Transcript: ENSMUST00000021458). Exon 6~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Sptb gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-177N17 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: Homozygotes for a spontaneous mutation exhibit a severe microcytic anemia with erythrocyte fragility, hepatomegaly, and jaundice. Mutants die within a few days of birth. Heterozygotes are mildly anemic.

Exon 6 starts from about 8.12% of the coding region. The knockout of Exon 6~8 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 637 bp, and the size of intron 8 for 3'-loxP site insertion: 746 bp. The size of effective cKO region: ~1594 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 4 5 6 7 8 9 36 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Sptb 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(8094bp) | A(24.18% 1957) | C(25.93% 2099) | T(26.08% 2111) | G(23.81% 1927)

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 - 76629226 76632225 3000 browser details YourSeq 122 2703 2891 3000 88.2% chr6 + 70940007 70940215 209 browser details YourSeq 118 2687 2881 3000 78.6% chr7 + 77533944 77534131 188 browser details YourSeq 118 2687 2886 3000 83.2% chr4 + 84296989 84297185 197 browser details YourSeq 117 2699 2892 3000 86.7% chr3 - 129883413 129883590 178 browser details YourSeq 117 2700 2881 3000 85.0% chr17 - 48764634 48764817 184 browser details YourSeq 117 2699 2890 3000 89.3% chr1 - 194326688 194376985 50298 browser details YourSeq 117 2703 2882 3000 84.2% chr10 + 28704430 28704629 200 browser details YourSeq 117 2703 2894 3000 79.7% chr1 + 35091686 35091874 189 browser details YourSeq 116 2700 2886 3000 89.4% chr5 + 151082192 151082397 206 browser details YourSeq 115 2699 2879 3000 83.6% chr12 - 71623035 71623241 207 browser details YourSeq 114 2699 2879 3000 91.4% chr13 + 107167595 107167785 191 browser details YourSeq 112 2687 2887 3000 84.4% chr17 - 80507148 80507345 198 browser details YourSeq 111 2687 2884 3000 81.8% chr7 + 82919789 82919984 196 browser details YourSeq 110 2699 2861 3000 84.5% chr8 - 49376080 49376239 160 browser details YourSeq 110 2728 2887 3000 91.7% chr12 - 91508273 91508436 164 browser details YourSeq 110 2699 2877 3000 82.9% chr1 - 190368651 190368820 170 browser details YourSeq 110 2704 2884 3000 89.8% chr3 + 127814021 127814218 198 browser details YourSeq 110 2699 2882 3000 83.9% chr3 + 104218013 104218193 181 browser details YourSeq 109 2699 2887 3000 89.3% chr14 - 55501918 55504925 3008

Note: The 3000 bp section upstream of Exon 6 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 - 76624632 76627631 3000 browser details YourSeq 169 2138 2346 3000 93.9% chr15 - 25275528 25275858 331 browser details YourSeq 166 2153 2347 3000 92.9% chr18 - 60981814 60982005 192 browser details YourSeq 166 2154 2348 3000 94.6% chr14 + 11994034 11994417 384 browser details YourSeq 165 2138 2333 3000 92.2% chr1 - 58429810 58429995 186 browser details YourSeq 163 2154 2348 3000 90.8% chr11 - 78570925 78571113 189 browser details YourSeq 162 2154 2333 3000 95.6% chr1 + 52112854 52113044 191 browser details YourSeq 161 2154 2348 3000 93.1% chr9 - 36715469 36715674 206 browser details YourSeq 160 2155 2348 3000 94.0% chr2 - 36052362 36052558 197 browser details YourSeq 158 2158 2402 3000 92.0% chr1 - 52635341 52635810 470 browser details YourSeq 158 2153 2333 3000 96.0% chr1 + 135285502 135285684 183 browser details YourSeq 157 2154 2348 3000 90.0% chr2 + 164511714 164511898 185 browser details YourSeq 156 2153 2338 3000 93.3% chr10 - 43558934 43559122 189 browser details YourSeq 156 2157 2347 3000 91.0% chr2 + 51454897 51455080 184 browser details YourSeq 153 2154 2333 3000 94.8% chr19 - 21119081 21119268 188 browser details YourSeq 153 2155 2333 3000 95.4% chr18 - 56628186 56628369 184 browser details YourSeq 153 2155 2348 3000 88.6% chr13 - 53810050 53810238 189 browser details YourSeq 153 2153 2346 3000 91.4% chr1 + 143622982 143623177 196 browser details YourSeq 152 2154 2341 3000 92.7% chr8 - 23313204 23313401 198 browser details YourSeq 152 2153 2333 3000 92.7% chr7 - 39923634 39923816 183

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Sptb beta, erythrocytic [ Mus musculus (house mouse) ] Gene ID: 20741, updated on 10-Oct-2019

Gene summary

Official Symbol Sptb provided by MGI Official Full Name spectrin beta, erythrocytic provided by MGI Primary source MGI:MGI:98387 See related Ensembl:ENSMUSG00000021061 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 ja; Spnb1; Gm1301; Spnb-1; AI842465; jaundiced; mKIAA4219; D330027P03Rik Expression Biased expression in cerebellum adult (RPKM 27.2), liver E14.5 (RPKM 17.4) and 13 other tissues See more Orthologs human all

Genomic context

Location: 12 C3; 12 33.73 cM See Sptb in Genome Data Viewer

Exon count: 36

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 12 NC_000078.6 (76580488..76710547, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 12 NC_000078.5 (77681475..77811534, complement)

Chromosome 12 - NC_000078.6

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Transcript information: This gene has 3 transcripts

Gene: Sptb ENSMUSG00000021061

Description spectrin beta, erythrocytic [Source:MGI Symbol;Acc:MGI:98387] Gene Synonyms D330027P03Rik, LOC383567, Spnb-1, Spnb1, brain erythroid spectrin (235E), spectrin R Location : 76,580,488-76,710,547 reverse strand. GRCm38:CM001005.2 About this gene This gene has 3 transcripts (splice variants), 206 orthologues, 36 paralogues, is a member of 1 Ensembl protein family and is associated with 53 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Sptb-201 ENSMUST00000021458.12 10394 2329aa ENSMUSP00000021458.6 Protein coding CCDS36477 Q3UGX2 TSL:1 GENCODE basic APPRIS P2

Sptb-202 ENSMUST00000166101.1 8084 2137aa ENSMUSP00000129782.1 Protein coding - E9Q397 TSL:5 GENCODE basic APPRIS ALT2

Sptb-203 ENSMUST00000170532.1 532 No protein - Retained intron - - TSL:1

150.06 kb Forward strand 76.60Mb 76.65Mb 76.70Mb Plekhg3-201 >protein coding Gm24010-201 >misc RNA (Comprehensive set...

Plekhg3-207 >protein coding

Plekhg3-208 >retained intron

Plekhg3-205 >retained intron

Contigs AC163033.4 > < AC132482.4 Genes (Comprehensive set... < Sptb-201protein coding

< Sptb-203retained intron

< Sptb-202protein coding

Regulatory Build

76.60Mb 76.65Mb 76.70Mb Reverse strand 150.06 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: ENSMUST00000021458

< Sptb-201protein coding

Reverse strand 130.06 kb

ENSMUSP00000021... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily CH domain superfamily SSF50729

SSF46966 SMART Spectrin/alpha-

Calponin homology domain Pleckstrin homology domain Prints Pleckstrin homology domain, spectrin-type Pfam Pleckstrin homology domain 9

Spectrin repeat PROSITE profiles Calponin homology domain Pleckstrin homology domain

PROSITE patterns Actinin-type -binding domain, conserved site

Actinin-type actin-binding domain, conserved site PIRSF Spectrin, beta subunit PANTHER PTHR11915:SF248

PTHR11915 Gene3D CH domain superfamily 1.20.58.1940 PH-like domain superfamily

1.20.58.60 CDD Calponin homology domain cd10571

cd00176

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

Variant Legend stop gained missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2329

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

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