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Mouse Carf Conditional Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Carf gene (NCBI Reference Sequence: NM_139150 ; Ensembl: ENSMUSG00000026017 ) is located on Mouse chromosome 1. 15 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 15 (Transcript: ENSMUST00000187978). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Carf gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-85G22 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: Mice homozygous for a null allele have aberrant learning and memory.

Exon 4 starts from about 14.71% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 1021 bp, and the size of intron 4 for 3'-loxP site insertion: 15401 bp. The size of effective cKO region: ~561 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 15 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Carf 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(7061bp) | A(25.07% 1770) | C(19.08% 1347) | T(36.71% 2592) | G(19.15% 1352)

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% chr1 + 60121602 60124601 3000 browser details YourSeq 520 1 1184 3000 86.5% chr7 - 127354823 127356075 1253 browser details YourSeq 508 1 1159 3000 86.0% chr9 - 35062140 35063346 1207 browser details YourSeq 352 1 856 3000 84.1% chr1 + 69876665 69877533 869 browser details YourSeq 340 1 621 3000 86.4% chr5 - 114838747 114839412 666 browser details YourSeq 312 1 498 3000 86.2% chr10 + 75490857 75491365 509 browser details YourSeq 310 345 1116 3000 85.1% chr19 + 6890474 6891372 899 browser details YourSeq 286 1336 1786 3000 84.5% chr18 + 34143521 34143985 465 browser details YourSeq 273 1 1852 3000 84.6% chr19 + 9139221 9399326 260106 browser details YourSeq 257 1240 1875 3000 81.2% chr6 + 149437670 149438260 591 browser details YourSeq 256 1240 1805 3000 84.4% chr9 - 74097941 74098598 658 browser details YourSeq 255 1335 1875 3000 83.9% chr8 + 75467358 75467946 589 browser details YourSeq 254 1419 1857 3000 83.0% chr1 - 131781296 131781738 443 browser details YourSeq 249 1276 1785 3000 83.9% chrX - 95499246 95499825 580 browser details YourSeq 243 1 479 3000 83.8% chr6 - 37488783 37489362 580 browser details YourSeq 238 44 492 3000 85.5% chr15 - 98444215 98444680 466 browser details YourSeq 228 1364 1862 3000 86.6% chrX_GL456233_random - 54735 55242 508 browser details YourSeq 225 1377 1830 3000 81.8% chr5 - 5829899 5830348 450 browser details YourSeq 223 1340 1700 3000 85.5% chr14 - 8560981 8561354 374 browser details YourSeq 221 1382 1841 3000 86.5% chr5 - 34071627 34072085 459

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% chr1 + 60125163 60128162 3000 browser details YourSeq 171 1450 1826 3000 91.0% chr11 - 75406948 75699039 292092 browser details YourSeq 162 1407 1837 3000 94.0% chr10 - 41750821 41819060 68240 browser details YourSeq 157 1435 1787 3000 88.0% chr4 - 45109623 45109934 312 browser details YourSeq 154 1404 1601 3000 93.8% chr7 - 127658303 127658729 427 browser details YourSeq 153 1379 1600 3000 86.0% chr12 - 97631129 97631327 199 browser details YourSeq 153 1418 1596 3000 94.3% chr11 - 57578884 57579261 378 browser details YourSeq 153 1423 1601 3000 93.8% chr10 - 56182391 56182611 221 browser details YourSeq 152 1416 1599 3000 92.2% chr10 - 128554424 128554776 353 browser details YourSeq 152 1451 1833 3000 91.9% chr8 + 72247912 72248294 383 browser details YourSeq 151 1301 1599 3000 85.3% chr11 + 86986545 86986722 178 browser details YourSeq 151 1437 1833 3000 91.9% chr1 + 162604687 162605080 394 browser details YourSeq 150 1416 1599 3000 90.6% chr11 - 30225195 30225377 183 browser details YourSeq 150 1439 1696 3000 91.2% chr11 - 5208119 5208397 279 browser details YourSeq 150 1419 1600 3000 92.7% chr1 - 165561908 165562101 194 browser details YourSeq 149 1408 1590 3000 93.2% chr15 + 45423416 45423634 219 browser details YourSeq 148 1032 1600 3000 92.1% chr1 - 143704588 143705191 604 browser details YourSeq 148 1466 1833 3000 89.8% chr15 + 81429292 81429686 395 browser details YourSeq 148 1422 1598 3000 92.5% chr1 + 39651466 39651656 191 browser details YourSeq 147 1432 1599 3000 94.7% chr7 - 129974050 129974221 172

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Carf calcium response factor [ Mus musculus (house mouse) ] Gene ID: 241066, updated on 12-Aug-2019

Gene summary

Official Symbol Carf provided by MGI Official Full Name calcium response factor provided by MGI Primary source MGI:MGI:2182269 See related Ensembl:ENSMUSG00000026017 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 Als2cr8; ECBRC-FC1; ECBRC-FC2 Expression Broad expression in testis adult (RPKM 2.5), CNS E18 (RPKM 1.9) and 25 other tissues See more Orthologs human all

Genomic context

Location: 1; 1 C2 See Carf in Genome Data Viewer

Exon count: 18

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (60098221..60153953)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (60155125..60207878)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 10 transcripts

Gene: Carf ENSMUSG00000026017

Description calcium response factor [Source:MGI Symbol;Acc:MGI:2182269] Gene Synonyms Als2cr8 Location Chromosome 1: 60,098,247-60,153,953 forward strand. GRCm38:CM000994.2 About this gene This gene has 10 transcripts (splice variants), 167 orthologues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Carf- ENSMUST00000187978.6 5463 689aa ENSMUSP00000141169.1 Protein coding CCDS14989 Q8VHI4 TSL:1 209 GENCODE basic APPRIS P3

Carf- ENSMUST00000180952.7 2157 689aa ENSMUSP00000137825.1 Protein coding CCDS14989 Q8VHI4 TSL:1 206 GENCODE basic APPRIS P3

Carf- ENSMUST00000027171.11 2102 654aa ENSMUSP00000027171.5 Protein coding CCDS78592 A8VI08 TSL:1 201 GENCODE basic APPRIS ALT2

Carf- ENSMUST00000124986.7 410 79aa ENSMUSP00000121293.1 Protein coding - D3Z2W4 CDS 3' 202 incomplete TSL:3

Carf- ENSMUST00000130075.7 2763 87aa ENSMUSP00000137867.1 Nonsense mediated - M0QWJ8 TSL:1 203 decay

Carf- ENSMUST00000186107.6 1837 255aa ENSMUSP00000139554.1 Nonsense mediated - A8VI09 TSL:1 207 decay

Carf- ENSMUST00000132949.2 493 55aa ENSMUSP00000139878.1 Nonsense mediated - A0A087WPQ8 CDS 5' 204 decay incomplete TSL:3

Carf- ENSMUST00000150008.7 3795 No - Retained intron - - TSL:2 205 protein

Carf- ENSMUST00000191232.1 3235 No - Retained intron - - TSL:NA 210 protein

Carf- ENSMUST00000186779.1 2416 No - Retained intron - - TSL:NA 208 protein

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75.71 kb Forward strand 60.10Mb 60.12Mb 60.14Mb 60.16Mb Genes Carf-203 >nonsense mediated decay (Comprehensive set...

Carf-202 >protein coding Carf-208 >retained intron

Carf-205 >retained intron Gm15464-201 >processed pseudogene Carf-204 >nonsense mediated decay

Carf-210 >retained intron

Carf-209 >protein coding

Carf-206 >protein coding

Carf-207 >nonsense mediated decay

Carf-201 >protein coding

Contigs AC116581.4 > < AC138597.7 Genes < Wdr12-201protein coding (Comprehensive set...

< Wdr12-202protein coding

< Wdr12-203protein coding

< Wdr12-206protein coding

< Wdr12-207protein coding

< Wdr12-204retained intron

Regulatory Build

60.10Mb 60.12Mb 60.14Mb 60.16Mb Reverse strand 75.71 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript pseudogene

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Transcript: ENSMUST00000187978

48.61 kb Forward strand

Carf-209 >protein coding

ENSMUSP00000141... MobiDB lite Low complexity (Seg) Pfam Calcium-responsive transcription factor PANTHER Calcium-responsive transcription factor

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

Variant Legend frameshift variant missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 600 689

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

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