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Mouse Chchd2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Chchd2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Chchd2 gene (NCBI Reference Sequence: NM_024166 ; Ensembl: ENSMUSG00000070493 ) is located on Mouse chromosome 5. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000094280). 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 Chchd2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-326M9 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 2 starts from about 11.11% 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: 3014 bp, and the size of intron 2 for 3'-loxP site insertion: 1354 bp. The size of effective cKO region: ~750 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 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Chchd2 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(7250bp) | A(23.99% 1739) | C(22.68% 1644) | T(29.05% 2106) | G(24.29% 1761)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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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% chr5 - 129884434 129887433 3000 browser details YourSeq 201 1350 1786 3000 90.1% chr18 - 60902538 60903146 609 browser details YourSeq 190 1350 1603 3000 92.9% chr11 - 113670879 113671227 349 browser details YourSeq 185 1350 1599 3000 92.3% chr11 - 77638951 77642925 3975 browser details YourSeq 173 1350 1775 3000 85.8% chr15 + 81517571 81517979 409 browser details YourSeq 168 1228 1525 3000 90.0% chr2 - 155693906 155694525 620 browser details YourSeq 166 1349 1594 3000 89.3% chr1 + 74673631 74674173 543 browser details YourSeq 164 1230 1520 3000 91.1% chr1 - 134484416 134485071 656 browser details YourSeq 163 1346 1649 3000 88.7% chr14 + 26857541 26858082 542 browser details YourSeq 159 1222 1516 3000 90.0% chr17 + 53532874 53533312 439 browser details YourSeq 155 1231 1525 3000 88.7% chr18 - 49712776 49877499 164724 browser details YourSeq 155 1239 1525 3000 91.1% chr14 + 7763242 7763850 609 browser details YourSeq 155 1228 1573 3000 87.5% chr11 + 107593046 107593507 462 browser details YourSeq 154 1322 1520 3000 89.4% chr7 + 25441261 25441882 622 browser details YourSeq 150 1349 1528 3000 92.3% chr18 - 36623361 36623708 348 browser details YourSeq 150 1347 1525 3000 93.2% chr10 - 116670759 116670942 184 browser details YourSeq 149 1222 1509 3000 81.5% chr4 - 41152465 41152712 248 browser details YourSeq 149 82 232 3000 99.4% chr4 + 34485727 34485877 151 browser details YourSeq 149 1350 1648 3000 87.6% chr11 + 76291425 76291988 564 browser details YourSeq 146 1348 1534 3000 90.7% chr5 - 150406005 150406202 198

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% chr5 - 129880684 129883683 3000 browser details YourSeq 485 883 1558 3000 97.5% chr5 + 129900123 129901036 914 browser details YourSeq 374 1102 2525 3000 92.4% chrX - 103762794 103763185 392 browser details YourSeq 365 1102 2525 3000 93.1% chr4 + 34486129 34486503 375 browser details YourSeq 360 1102 2523 3000 92.8% chr4 - 148067820 148068195 376 browser details YourSeq 293 1102 2525 3000 87.1% chr15 + 63760442 63760816 375 browser details YourSeq 292 1114 2525 3000 86.2% chr4 - 6507714 6508090 377 browser details YourSeq 286 1102 2525 3000 88.5% chrX - 142533263 142533603 341 browser details YourSeq 262 1102 2510 3000 85.5% chr11 - 6650181 6650539 359 browser details YourSeq 186 2301 2525 3000 92.7% chr7 + 28000121 28000347 227 browser details YourSeq 167 2287 2525 3000 89.2% chr16 + 30255536 30255785 250 browser details YourSeq 162 2301 2525 3000 95.1% chr13 - 26899241 26899465 225 browser details YourSeq 143 1421 1581 3000 92.4% chr9 - 65599551 65599706 156 browser details YourSeq 137 1421 1580 3000 91.0% chr7 - 19122464 19122619 156 browser details YourSeq 130 1432 1581 3000 95.9% chr11 + 102191689 102191851 163 browser details YourSeq 126 1437 1581 3000 96.4% chr1 + 72649951 72650096 146 browser details YourSeq 121 1455 1583 3000 96.9% chr4 - 59559698 59559826 129 browser details YourSeq 118 1450 1581 3000 94.7% chr8 - 3561153 3561284 132 browser details YourSeq 116 1449 1580 3000 94.0% chr1 + 145403637 145403768 132 browser details YourSeq 108 1459 1581 3000 94.4% chr10 - 76390754 76390878 125

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: Chchd2 coiled-coil-helix-coiled-coil-helix domain containing 2 [ Mus musculus (house mouse) ] Gene ID: 14004, updated on 21-Aug-2019

Gene summary

Official Symbol Chchd2 provided by MGI Official Full Name coiled-coil-helix-coiled-coil-helix domain containing 2 provided by MGI Primary source MGI:MGI:1261428 See related Ensembl:ENSMUSG00000070493 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 Etohi6; AL033347 Expression Ubiquitous expression in adrenal adult (RPKM 1404.4), duodenum adult (RPKM 1158.2) and 28 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 G1.3 See Chchd2 in Genome Data Viewer

Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (129881161..129887470, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (130357032..130363340, complement)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 2 transcripts

Gene: Chchd2 ENSMUSG00000070493

Description coiled-coil-helix-coiled-coil-helix domain containing 2 [Source:MGI Symbol;Acc:MGI:1261428] Gene Synonyms Etohi6 Location Chromosome 5: 129,881,156-129,887,470 reverse strand. GRCm38:CM000998.2 About this gene This gene has 2 transcripts (splice variants), 294 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Chchd2-201 ENSMUST00000094280.3 915 153aa ENSMUSP00000091835.3 Protein coding CCDS19702 Q9D1L0 TSL:1 GENCODE basic APPRIS P1

Chchd2-202 ENSMUST00000131645.1 2069 No protein - Retained intron - - TSL:2

26.32 kb Forward strand 129.875Mb 129.880Mb 129.885Mb 129.890Mb 129.895Mb Genes Zbed5-203 >retained intron (Comprehensive set...

Zbed5-201 >protein coding

Zbed5-202 >protein coding

Contigs < AC242408.2 AC164071.3 > Genes (Comprehensive set... < Phkg1-201protein coding < Chchd2-201protein coding

< Phkg1-204protein coding < Gm42790-201TEC < Chchd2-202retained intron

< Phkg1-202protein coding

< Phkg1-205lncRNA < Phkg1-206retained intron

Regulatory Build

129.875Mb 129.880Mb 129.885Mb 129.890Mb 129.895Mb Reverse strand 26.32 kb

Regulation Legend

CTCF Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000094280

< Chchd2-201protein coding

Reverse strand 6.32 kb

ENSMUSP00000091... MobiDB lite Low complexity (Seg) PROSITE profiles PS51808 PANTHER PTHR13523:SF3

PTHR13523

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

Variant Legend

synonymous variant

Scale bar 0 20 40 60 80 100 120 153

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

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