https://www.alphaknockout.com

Mouse Ccdc186 Knockout Project (CRISPR/Cas9)

Objective: To create a Ccdc186 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ccdc186 (NCBI Reference Sequence: NM_170757 ; Ensembl: ENSMUSG00000035173 ) is located on Mouse 19. 15 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 15 (Transcript: ENSMUST00000076085). Exon 2~8 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note:

Exon 2 starts from about 25.08% of the coding region. Exon 2~8 covers 31.99% of the coding region. The size of effective KO region: ~9453 bp. The KO 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 2 3 4 5 6 7 8 15

Legends Exon of mouse Ccdc186 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

Overview of the Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 706 bp section downstream of Exon 8 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(23.95% 479) | C(22.8% 456) | T(33.8% 676) | G(19.45% 389)

Note: The 2000 bp section upstream of Exon 2 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(706bp) | A(23.65% 167) | C(23.09% 163) | T(29.46% 208) | G(23.8% 168)

Note: The 706 bp section downstream of Exon 8 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 2000 1 2000 2000 100.0% chr19 - 56810366 56812365 2000 browser details YourSeq 37 1272 1322 2000 95.2% chr8 - 21060144 21060649 506 browser details YourSeq 27 1258 1295 2000 96.7% chr3 - 109941696 109941733 38 browser details YourSeq 26 1230 1264 2000 75.9% chr17 - 82589481 82589511 31 browser details YourSeq 26 1270 1302 2000 96.5% chr1 - 37744249 37744392 144 browser details YourSeq 25 898 926 2000 81.5% chr1 - 61076235 61076261 27 browser details YourSeq 24 301 324 2000 100.0% chr16 + 34402490 34402513 24 browser details YourSeq 23 1747 1771 2000 87.5% chr7 + 18522969 18522992 24 browser details YourSeq 23 883 905 2000 100.0% chr6 + 64388151 64388173 23

Note: The 2000 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 706 1 706 706 100.0% chr19 - 56800207 56800912 706 browser details YourSeq 30 87 129 706 94.2% chr11 - 37374336 37374387 52 browser details YourSeq 29 157 413 706 45.2% chr11 + 74144996 74145029 34 browser details YourSeq 25 58 101 706 66.7% chr14 - 94944961 9