https://www.alphaknockout.com

Mouse Plekhg2 Knockout Project (CRISPR/Cas9)

Objective: To create a Plekhg2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Plekhg2 (NCBI Reference Sequence: NM_138752 ; Ensembl: ENSMUSG00000037552 ) is located on Mouse 7. 19 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 19 (Transcript: ENSMUST00000094644). Exon 3~16 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 3 starts from about 2.73% of the coding region. Exon 3~16 covers 34.43% of the coding region. The size of effective KO region: ~6200 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' 11 13 15

1 3 4 5 6 7 8 9 10 12 14 16 19

Legends Exon of mouse Plekhg2 Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 617 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 1298 bp section downstream of Exon 16 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.

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

Sequence 12

Summary: Full Length(617bp) | A(25.77% 159) | C(19.12% 118) | T(21.39% 132) | G(33.71% 208)

Note: The 617 bp section upstream of Exon 3 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(1298bp) | A(29.35% 381) | C(20.11% 261) | T(23.73% 308) | G(26.81% 348)

Note: The 1298 bp section downstream of Exon 16 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 617 1 617 617 100.0% chr7 - 28370498 28371114 617 browser details YourSeq 24 320 347 617 85.2% chr15 + 58592031 58592057 27 browser details YourSeq 22 124 147 617 87.0% chr10 - 103873805 103873827 23 browser details YourSeq 22 278 303 617 84.0% chr14 + 42356176 42356200 25 browser details YourSeq 22 278 303 617 84.0% chr14 + 41216411 41216435 25 browser details YourSeq 21 450 474 617 92.0% chr16 - 21924961 21924985 25 browser details YourSeq 20 100 119 617 100.0% chr10 - 111361649 111361668 20 browser details YourSeq 20 231 250 617 100.0% chr13 + 37288190 37288209 20

Note: The 617 bp section upstream of Exon 3 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 1298 1 1298 1298 100.0% chr7 - 28363000 28364297 1298 browser details YourSeq 218 443 737 1298 90.9% chr7 - 79356716 79357047 332 browser details YourSeq 204 434 741 1298 92.9% chr17 + 24523547 24523959 413 browser details YourSeq 190 451 744 1298 90.3% chr1 - 118434448 118434764 317 browser details YourSeq 189 458 728 1298