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Mouse Xirp2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Xirp2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Xirp2 gene (NCBI Reference Sequence: NM_001083919 ; Ensembl: ENSMUSG00000027022 ) is located on Mouse chromosome 2. 9 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 8 (Transcript: ENSMUST00000112347). Exon 6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Xirp2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-93G17 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: Homozygous null mice exhibit severe growth retardation, abnormal myocardial fiber morphology, failure of intercalated disc maturation, cardiac conduction and ventricular septal defects, altered ionic currents in cardiomyocytes, and postnatal lethality.

Exon 6 starts from about 3.66% of the coding region. The knockout of Exon 6 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 1471 bp, and the size of intron 6 for 3'-loxP site insertion: 728 bp. The size of effective cKO region: ~634 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 4 5 6 7 9 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Xirp2 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(7134bp) | A(34.05% 2429) | C(18.53% 1322) | T(28.5% 2033) | G(18.92% 1350)

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% chr2 + 67503798 67506797 3000 browser details YourSeq 173 8 279 3000 85.1% chr10 - 45038097 45038374 278 browser details YourSeq 164 7 293 3000 88.7% chr16 + 91123764 91124064 301 browser details YourSeq 163 1 282 3000 86.3% chr2 - 27150590 27150869 280 browser details YourSeq 162 1 292 3000 85.3% chr3 - 31752645 31752937 293 browser details YourSeq 161 1 286 3000 83.9% chr3 + 51366355 51366645 291 browser details YourSeq 157 4 292 3000 83.4% chr3 - 129081506 129081794 289 browser details YourSeq 157 9 290 3000 87.6% chr1 + 120525237 120525522 286 browser details YourSeq 156 5 292 3000 86.8% chr1 - 189950160 189950449 290 browser details YourSeq 156 8 291 3000 87.9% chr1 - 156497642 156497940 299 browser details YourSeq 154 1 292 3000 86.4% chr1 + 78516238 78516530 293 browser details YourSeq 152 64 282 3000 91.9% chr7 + 90464051 90526223 62173 browser details YourSeq 152 8 290 3000 83.2% chr6 + 115876297 115876566 270 browser details YourSeq 150 14 292 3000 90.0% chr18 + 4631283 4631559 277 browser details YourSeq 149 15 291 3000 87.4% chr7 - 82408742 82409018 277 browser details YourSeq 148 1 293 3000 86.0% chr8 - 18644041 18644333 293 browser details YourSeq 147 4 279 3000 85.4% chr17 - 88092183 88092466 284 browser details YourSeq 144 1 292 3000 87.8% chr15 + 76316690 76316983 294 browser details YourSeq 144 42 292 3000 80.8% chr13 + 12049731 12049962 232 browser details YourSeq 142 14 292 3000 88.6% chr7 - 93127415 93127693 279

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% chr2 + 67507432 67510431 3000 browser details YourSeq 31 182 241 3000 78.2% chr1 + 54963731 54963798 68 browser details YourSeq 25 171 196 3000 100.0% chr1 + 185975331 185975671 341 browser details YourSeq 23 1198 1226 3000 89.7% chr4 + 53825876 53825904 29 browser details YourSeq 23 204 230 3000 92.6% chr12 + 3712956 3712982 27 browser details YourSeq 21 216 236 3000 100.0% chr7 - 6397899 6397919 21 browser details YourSeq 20 204 229 3000 88.5% chr1 + 112632260 112632285 26

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Xirp2 xin actin-binding repeat containing 2 [ Mus musculus (house mouse) ] Gene ID: 241431, updated on 12-Aug-2019

Gene summary

Official Symbol Xirp2 provided by MGI Official Full Name xin actin-binding repeat containing 2 provided by MGI Primary source MGI:MGI:2685198 See related Ensembl:ENSMUSG00000027022 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 Xin2; Cmya3; Gm352; AI452089; 2310003D02Rik; 2310008C07Rik; A530024P18Rik Expression Biased expression in heart adult (RPKM 8.8) and mammary gland adult (RPKM 2.5) See more Orthologs human all

Genomic context

Location: 2; 2 C1.3 See Xirp2 in Genome Data Viewer

Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (67446000..67526620)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (67284059..67364663)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 11 transcripts

Gene: Xirp2 ENSMUSG00000027022

Description xin actin-binding repeat containing 2 [Source:MGI Symbol;Acc:MGI:2685198] Gene Synonyms 2310003D02Rik, 2310008C07Rik, A530024P18Rik, Cmya3, mXin beta, myomaxin Location Chromosome 2: 67,173,834-67,526,615 forward strand. GRCm38:CM000995.2 About this gene This gene has 11 transcripts (splice variants), 241 orthologues, 1 paralogue, is a member of 2 Ensembl protein families and is associated with 36 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Xirp2-202 ENSMUST00000112347.7 11979 3283aa ENSMUSP00000107966.1 Protein coding CCDS38133 Q4U4S6 TSL:1 GENCODE basic

Xirp2-201 ENSMUST00000028410.3 11955 3784aa ENSMUSP00000028410.3 Protein coding CCDS16082 Q4U4S6 TSL:1 GENCODE basic APPRIS P1

Xirp2-205 ENSMUST00000238912.1 12612 3512aa ENSMUSP00000159084.1 Protein coding - - GENCODE basic

Xirp2-207 ENSMUST00000239009.1 3347 930aa ENSMUSP00000158994.1 Protein coding - - GENCODE basic

Xirp2-209 ENSMUST00000239060.1 2704 701aa ENSMUSP00000159181.1 Protein coding - B2BBR6 GENCODE basic

Xirp2-204 ENSMUST00000238878.1 1095 284aa ENSMUSP00000158861.1 Protein coding - - CDS 3' incomplete

Xirp2-211 ENSMUST00000239164.1 754 119aa ENSMUSP00000159177.1 Protein coding - - CDS 5' incomplete

Xirp2-203 ENSMUST00000134620.2 1966 No protein - Retained intron - - TSL:1

Xirp2-210 ENSMUST00000239121.1 2935 No protein - lncRNA - - -

Xirp2-208 ENSMUST00000239053.1 2908 No protein - lncRNA - - -

Xirp2-206 ENSMUST00000238989.1 2860 No protein - lncRNA - - -

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372.78 kb Forward strand 67.2Mb 67.3Mb 67.4Mb 67.5Mb Genes (Comprehensive set... Xirp2-205 >protein coding

Xirp2-207 >protein coding

Xirp2-203 >retained intron Gm21830-201 >protein coding Xirp2-211 >protein coding

Xirp2-208 >lncRNA

Xirp2-206 >lncRNA

Xirp2-210 >lncRNA

Gm23328-201 >snoRNA

Xirp2-202 >protein coding

Xirp2-201 >protein coding

Xirp2-209 >protein coding

Xirp2-204 >protein coding

Contigs AL845473.7 > AL773549.5 > AL929411.11 >

Genes < Gm13598-201lncRNA < Gm13599-201lncRNA < Gm13601-201lncRNA (Comprehensive set...

Regulatory Build

67.2Mb 67.3Mb 67.4Mb 67.5Mb Reverse strand 372.78 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000112347

80.61 kb Forward strand

Xirp2-202 >protein coding

ENSMUSP00000107... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Actin-binding, Xin repeat PROSITE profiles Actin-binding, Xin repeat PANTHER Xin actin-binding repeat-containing protein 1/2

Xin actin-binding repeat-containing protein 2

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

Variant Legend inframe insertion inframe deletion missense variant splice region variant synonymous variant

Scale bar 0 400 800 1200 1600 2000 2400 2800 3283

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

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