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Mouse Kifap3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Kifap3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Kifap3 gene (NCBI Reference Sequence: NM_010629 ; Ensembl: ENSMUSG00000026585 ) is located on Mouse chromosome 1. 21 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 20 (Transcript: ENSMUST00000077642). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Kifap3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-87C19 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: About 70% of homozygotes for a knock-out mutation die of heart failure shortly after birth due to massive cardiomyocyte apoptosis triggered by cardiovascular overload. Neonatal thymocytes and developing neuronal cells undergo apoptosis while cultured thymocytes are susceptible to apoptotic inducers.

Exon 5 starts from about 16.23% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1211 bp, and the size of intron 5 for 3'-loxP site insertion: 16975 bp. The size of effective cKO region: ~642 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 21 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Kifap3 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(7142bp) | A(26.35% 1882) | C(19.84% 1417) | T(35.14% 2510) | G(18.66% 1333)

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 + 163794003 163797002 3000 browser details YourSeq 325 2416 3000 3000 87.5% chr15 + 73560151 73560743 593 browser details YourSeq 316 2390 2984 3000 86.9% chr12 - 109816099 109816689 591 browser details YourSeq 313 2390 3000 3000 87.6% chrX - 159603072 159603677 606 browser details YourSeq 303 2482 2994 3000 86.4% chr10 - 42879877 42880387 511 browser details YourSeq 303 2437 2991 3000 89.1% chr8 + 86462866 86463418 553 browser details YourSeq 302 2371 3000 3000 85.6% chr3 - 66040004 66040623 620 browser details YourSeq 302 2416 3000 3000 84.6% chr1 + 135238642 135239239 598 browser details YourSeq 301 2391 3000 3000 87.7% chr3 + 100698627 100699251 625 browser details YourSeq 296 2424 2957 3000 87.4% chr6 - 135648879 135649443 565 browser details YourSeq 295 2426 2995 3000 85.7% chr3 + 82353471 82354056 586 browser details YourSeq 295 2491 2975 3000 87.3% chr11 + 62895700 62896171 472 browser details YourSeq 289 2437 3000 3000 85.2% chr11 - 5519130 5519700 571 browser details YourSeq 289 2428 2958 3000 86.6% chr1 - 59072346 59073103 758 browser details YourSeq 286 2416 2998 3000 83.2% chr16 - 8020159 8020746 588 browser details YourSeq 286 2491 2983 3000 87.1% chr11 - 35715287 35715777 491 browser details YourSeq 281 2442 2997 3000 86.5% chr11 + 52169170 52169732 563 browser details YourSeq 279 2411 3000 3000 89.0% chrX - 163195452 163196040 589 browser details YourSeq 278 2506 3000 3000 87.4% chr1 + 121775723 122117148 341426 browser details YourSeq 275 2411 3000 3000 86.5% chr19 + 60922153 60922742 590

Note: The 3000 bp section upstream of Exon 5 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 + 163797645 163800644 3000 browser details YourSeq 1276 1540 3000 3000 94.7% chr19 - 61128221 61129700 1480 browser details YourSeq 1272 1531 3000 3000 94.5% chr11 - 57363884 57365371 1488 browser details YourSeq 1271 1531 2991 3000 95.2% chr8 - 41520815 41522283 1469 browser details YourSeq 1269 1523 3000 3000 94.3% chr2 - 21285518 21287000 1483 browser details YourSeq 1268 1523 2998 3000 94.3% chr4 + 65676556 65678036 1481 browser details YourSeq 1267 1536 3000 3000 94.6% chr6 - 84663356 84664828 1473 browser details YourSeq 1265 1536 3000 3000 94.2% chr9 - 46800795 46802257 1463 browser details YourSeq 1265 1532 3000 3000 93.6% chr3 + 118147844 118149322 1479 browser details YourSeq 1264 1533 3000 3000 93.9% chr4 - 101679865 101681322 1458 browser details YourSeq 1264 1530 3000 3000 94.2% chr12 - 12151528 12152996 1469 browser details YourSeq 1264 1534 3000 3000 94.1% chr1 - 127071851 127073317 1467 browser details YourSeq 1264 1556 3000 3000 94.6% chr9 + 104068060 104069491 1432 browser details YourSeq 1264 1528 3000 3000 94.4% chr2 + 129350421 129351903 1483 browser details YourSeq 1264 1531 3000 3000 94.2% chr11 + 61033984 61035452 1469 browser details YourSeq 1264 1528 3000 3000 94.5% chr10 + 25792675 25794167 1493 browser details YourSeq 1262 1519 3000 3000 93.6% chr8 - 31052735 31054216 1482 browser details YourSeq 1262 1527 3000 3000 93.5% chr14 - 66426989 66428458 1470 browser details YourSeq 1259 1533 3000 3000 93.8% chrX - 36505327 36506790 1464 browser details YourSeq 1257 1517 3000 3000 93.4% chr2 - 94988071 94989567 1497

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 protein information: Kifap3 kinesin-associated protein 3 [ Mus musculus (house mouse) ] Gene ID: 16579, updated on 10-Oct-2019

Gene summary

Official Symbol Kifap3 provided by MGI Official Full Name kinesin-associated protein 3 provided by MGI Primary source MGI:MGI:107566 See related Ensembl:ENSMUSG00000026585 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as KAP3; SMAP; KAP-3 Summary The protein encoded by this gene is the non-motor subunit of kinesin-2 complex, and forms a heterotrimer with two Expression members of the kinesin superfamily of proteins that together form a microtubule plus-end directed translocator that plays an important role in intracellular transport, mitosis, and cell-cell adhesion. This protein contains multiple armadillo repeats involved in protein binding, and may serve as an adaptor to regulate binding of cargo with the motor proteins. Conditional disruption of this gene in mouse neural precursor cells caused a tumor-like phenotype and defective organization of the neuroepithelium thought to be the result of altered N-cadherin subcellular localization. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Mar 2015] Orthologs Broad expression in CNS E18 (RPKM 36.2), cortex adult (RPKM 29.0) and 15 other tissues See more human all

Genomic context

Location: 1; 1 H2.1 See Kifap3 in Genome Data Viewer Exon count: 22

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (163762138..163917107)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (165709810..165847231)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 2 transcripts

Gene: Kifap3 ENSMUSG00000026585

Description kinesin-associated protein 3 [Source:MGI Symbol;Acc:MGI:107566] Gene Synonyms KAP3, Smg GDS Location Chromosome 1: 163,779,583-163,917,109 forward strand. GRCm38:CM000994.2 About this gene This gene has 2 transcripts (splice variants), 240 orthologues, is a member of 1 Ensembl protein family and is associated with 27 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Kifap3-202 ENSMUST00000077642.11 4012 772aa ENSMUSP00000076830.5 Protein coding CCDS35751 P70188 TSL:1 GENCODE basic

Kifap3-201 ENSMUST00000027877.6 3946 793aa ENSMUSP00000027877.5 Protein coding CCDS78729 P70188 TSL:1 GENCODE basic APPRIS P1

157.53 kb Forward strand 163.80Mb 163.85Mb 163.90Mb Genes (Comprehensive set... Kifap3-201 >protein coding

Kifap3-202 >protein coding

Contigs < AC163899.3 < AC102444.8 Regulatory Build

163.80Mb 163.85Mb 163.90Mb Reverse strand 157.53 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

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Transcript: ENSMUST00000077642

137.53 kb Forward strand

Kifap3-202 >protein coding

ENSMUSP00000076... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Armadillo-type fold SMART Armadillo

SM01297 Pfam PF05804 PROSITE profiles Armadillo PANTHER Kinesin-associated protein 3 Gene3D Armadillo-like helical

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

Variant Legend frameshift variant missense variant synonymous variant

Scale bar 0 80 160 240 320 400 480 560 640 772

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

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