Question

I was assigned a gene: MAP65-2. I need to find the following:

• Known/reported function

  o Gene structure (exon/intron)

  o Primers (location, sequences, product size)

• Phylogenetic tree (known homologs)

• Gene expression pattern

• Guide RNA sequence

  o Primers for gRNA construct (location, sequences)

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if you can try solving this it would be greatly appreciated

Answer 1

Solution

Gene Name : MAP65-2

Organism: Arabidopsis thaliana

Protein: 65kDa microtubule associated protein 2.

Function: This protein is known to stabilize microtubules (MT). It regulates MT organization and dynamics. It provides microtubular resistance to drug propyzamide.

Gene Structure

MAP65-2

Gene nucleotide sequence:

GAAAAGACAGGGTCTCTCACCTTTTTGCTGTTGTTTTTTCCTCATTAATTATTGAGTTCCGTAAAATGGG TCCCACTTTTCCCATTTCCCAACCTCCATAACCAAAGTTGGAGATTTCTCTGTTTCCTATGCTCTCTCTC TCTCTTACTCTCACAGTGACTATTCGTCGCTTCAGATCTAAAAGAGAGGAAGATAAACCATTTGGATTCA ATATCGATTAAGGTAAGAAAGCCATCTTCTACTTGTTGGATTCGATTAGTTTCATTTCAGCTCATTGCCT CTTTACTTCTTCAATGTTGATTTCTTGCTTTGGCTTTGGTTTTATCACCAATGCTGTTTGATTTCTTTGA AGTTCCTTTCGAGCTTTTCTTGAGCTTAGTTTCACATTCTTAGCTTCACTAAGTTCTTTGATGTGTTTTT GTTCCCAAGTTCAAAATGTTGTTTCTGATTCAATTTGGTTTTCTAAAAAAGATTGCAACTTTGGTTTCTT TTAGTGTATAGTTAGTTTGGATCTTCACTAAATATGTTCGTTGTTTCTACTTTTGTAGGAAACCTTTTGA CTTCACAAGAATGGCAGTGACAGAAGCAGAAAATCCTCTTCTTGGTGAAATCACTTGTGGCACCTTACTA CAGAAGTTGCAGGTAGATTTCGCAACATACACACTTATCCTTTTAATATATGGTTCCACAAACTCAATGT ACAAACTTGTTACAGGAAATCTGGGATGAGGTTGGGGAGAGTGATGAAGAACGAGACAAACTGCTTCTAC AGATTGAGGAAGAGTGTCTGAATGTTTACAAAAAGAAAGTTGAGCTAGCCGCAAAATCTCGTGCAGAGCT TCTTCAGACTTTGTCAGATGCCACTGTTGAACTTTCCAATCTCACAACTGCTCTCGGGGAAAAAAGCTAT ATCGACATTGTAAGTAATCTCACGTCTTACAAGTGGTAAAGAGATGAGATGGTTACAGTGTAGTGTGATT CAATGTCTTTGTTTCCATTGCAGCCTGATAAGACTTCAGGAACGATCAAAGAACAACTTTCTGCAATAGC ACCTGCGCTTGAACAACTTTGGCAACAGAAAGAGGAAAGGGTCCGGGCATTTTCTGATGTACAATCACAG ATTCAGAAGATATGTGAGGAGATCGCTGGCGGATTGAACAATGGACCTCATGTGGTTGATGAGACTGACT TGTCTCTAAAGAGATTAGATGACTTCCAGAGGAAACTCCAAGAGCTCCAGAAAGAGAAGGTAAAATATTA GTTGGGATATGGTTATGTGTGGCCTATGTTCAAACCAGAACTAAAACTGCTTTGTGTTTGAGCAGAGTGA TAGGTTACAGAAGGTGCTCGAGTTTGTGAGCACGGTGCATGATCTATGTGCCGTCCTTCGTTTAGATTTC TTAAGCACTGTCACCGAAGTTCATCCGAGCTTAGATGAGGCAAATGGTGTTCAAACCAAGAGCATTAGCA ATGAAACACTTGCAAGGTTGGCTAAGACTGTCTTGACTCTTAAAGAAGATAAAATGCAAAGACTTAAGAA GGTTAGTCAAAATCATGGTCTTTGTGATCTCTGACATGAGCTTGGTCAGTTTAGGCTGCACATATGTTGA AATCCTTTCTGATTAATTGCGCTGAAAGTTTGTAACTTTTCTCTGCACCATTCTTGTTTGTGGATTTCAT TAATGTATCTTGGTTTTATGCAGCTTCAAGAACTAGCTACTCAGCTAACTGATCTATGGAATCTGATGGA TACTTCTGATGAGGAAAGGGAGCTTTTTGACCATGTTACCTCTAACATTTCAGCTTCAGTTCATGAAGTG ACTGCCTCTGGTGCTCTCGCACTTGATCTTATCGAACAGGTATCGTAAATTTGTAATGTATCAATCAATG TGTATGATAAGTACTGCTTTTAAAAATCTTCTGTGAACTCTTGAAGGCTGAGGTGGAAGTGGATAGGCTT GATCAACTGAAATCTAGCAGAATGAAGGAAATTGCATTCAAAAAGCAATCAGAGCTTGAGGAGATATATG CTCGTGCTCACATAGAAATAAAACCAGAGGTTGTTCGTGAGAGGATCATGTCGTTGATTGATGCTGGGAA TACTGAACCTACTGAGTTACTGGCTGACATGGATAGCCAGATCGCAAAGGCCAAGGAGGAAGCCTTTAGT AGAAAAGAAATTTTAGACCGTGTAGAGAAATGGATGTCAGCTTGTGAGGAAGAGAGCTGGCTCGAAGATT ACAATCGAGTATGCTAACATTGGCTATGTTAATAAACACATTACTTATTAAGCATAGAGTTCAATTGATG ATTCTTTTGGGTTTTTGTTCTGGAAAAACAGGATCAGAACAGGTATAGTGCAAGCCGAGGTGCGCATTTG AATCTCAAGAGAGCTGAGAAAGCTCGGATTCTGGTCAGCAAGATTACTGGTAAGAACTTCCAAAAGTCTC TCTTAGTTATTACTACACATAGAATTAAACTGAGACTGAAGAATATATCATACTTTGTTTGTGCAAGCTA TGGTTGACACGTTGATTGCTAAAACCCGGGCATGGGAAGAGGAGAACAGTATGTCCTTTGAGTACGATGG AGTTCCTTTACTCGCCATGCTAGATGAATACACTATGCTTAGGCAAGAACGAGAAGATGAGAAGCGGAGG CTGAAAGTAAGTTGTTTCATCCTGTGTTAAACCGATTGTTGAAACTAACTTCTTTTAAAATAATCTTGCC TTTTCATTAACAGGAACAGAAGAAGCAGCAAGAACAGCCACACACAGATCAAGAATCCGCCTTTGGGTCT AAACCAAGCCCTGCAAGACCCGTTAGTGCCAAGAAACCGGTGGGAACACGAGTCAATGGAGGAGGGCTCA ACGAAACTCCTATGAGGCGTTTGTCTATGAACTCAAACCAGAACGGAAGCAAGTCCAAGAGAGACAGTCT GAACAAGATAGCTTCACCTTCGAACATTGTAGCCAACACAAAAGATGATGCTGCATCTCCAGTCTCTCGT GCTGACCCAGTGATGGCTTCACCGTGACAATATCAAAAGAAAGAGAGTTATAATGCTTACTTACACTTTT GTTAGTTGCTAATAGTGCTGTTTAAGAAACAAACACTTAGAAACAAAGACAATGAGAGTAGAATGTTACT TAGATGTTTCTGTTTCTGTATTCATTCACTCAAATTTGTTTGGTACTTGTTAGCATGACTAATGTGAAGT GACCGGTTTAGTGAATGAAAGTCAAGGTTCTAATTGGAATTACGAAATAATATTTGGGC

Exons represented as green = 10

Introns represented as lines and arrows = 9

Primers

Forward primer: GAAAAGACAGGGTCTCTCACCTTTTTGC [Tm 62oC]

Location: +1 to +28 of the gene

Reverse primer: GCCCAAATATTATTTCGTAATTCCAATTAGAACCTTGAC [Tm 60oC]

Location: +3240 to +3279 of the gene

PCR Product size = 3.279 kb

Phylogenetic tree

AtMAP65-1 100 51 ADMAP65-2 100 Os06g40840 100 Os06g20370 Oso2g48830 Oso5g33890 100 77 AtMAP65-5 Os08g41890 100 AtMAP65-8 95 64 Os09927700 Os03g13460 92 100 AtMAP65-6 100 AtMAP65-7 Os03g50970 100 Os01949200 Os05947970 94 AtMAP65-3 79 80 ADMAP65-9 99 Os02g03400 AtMAP65-4 ScAse1P 100 100 SpAse1 HSPRC1

Known most similar homolog : Arabidopsis thaliana MAP65-1

Other homologs belong to Oryza sativa, Saccharomyces cerevisiae (ScAse1p), Schizosaccharomyces pombe (SpAse1) and human (HsPRC1).

Gene expression pattern

TOO Seed endosperm plant embryo 4 Liu et al Pignatta et al., 2014 - Organismi Pignatta et al., 2014 - Organism Pignatta et al., 2014 - Organism Pignatta et al., 2014 - Organism Pignatta et al., 2014 - Organismi Pignatta et al., 2014 - Organism Pignatta et al., 2014 - Organismi Pignatta et al., 2014 - Organism Pignatta et al., 2014 - Organism

According to the experiments perfomed all over the world the gene MAP65-2 has been seen to be differentially expressed in: endosperm, plant embryo, seed, flower, fruit, leaf and roots.

Guide RNA Sequence

mRNA sequence (excluding introns)

GAAAAGACAGGGTCTCTCACCTTTTTGCTGTTGTTTTTTCCTCATTAATTATTGAGTTCCGTAAAATGGG TCCCACTTTTCCCATTTCCCAACCTCCATAACCAAAGTTGGAGATTTCTCTGTTTCCTATGCTCTCTCTC TCTCTTACTCTCACAGTGACTATTCGTCGCTTCAGATCTAAAAGAGAGGAAGATAAACCATTTGGATTCA ATATCGATTAAGGAAACCTTTTGACTTCACAAGAATGGCAGTGACAGAAGCAGAAAATCCTCTTCTTGGT GAAATCACTTGTGGCACCTTACTACAGAAGTTGCAGGAAATCTGGGATGAGGTTGGGGAGAGTGATGAAG AACGAGACAAACTGCTTCTACAGATTGAGGAAGAGTGTCTGAATGTTTACAAAAAGAAAGTTGAGCTAGC CGCAAAATCTCGTGCAGAGCTTCTTCAGACTTTGTCAGATGCCACTGTTGAACTTTCCAATCTCACAACT GCTCTCGGGGAAAAAAGCTATATCGACATTCCTGATAAGACTTCAGGAACGATCAAAGAACAACTTTCTG CAATAGCACCTGCGCTTGAACAACTTTGGCAACAGAAAGAGGAAAGGGTCCGGGCATTTTCTGATGTACA ATCACAGATTCAGAAGATATGTGAGGAGATCGCTGGCGGATTGAACAATGGACCTCATGTGGTTGATGAG ACTGACTTGTCTCTAAAGAGATTAGATGACTTCCAGAGGAAACTCCAAGAGCTCCAGAAAGAGAAGAGTG ATAGGTTACAGAAGGTGCTCGAGTTTGTGAGCACGGTGCATGATCTATGTGCCGTCCTTCGTTTAGATTT CTTAAGCACTGTCACCGAAGTTCATCCGAGCTTAGATGAGGCAAATGGTGTTCAAACCAAGAGCATTAGC AATGAAACACTTGCAAGGTTGGCTAAGACTGTCTTGACTCTTAAAGAAGATAAAATGCAAAGACTTAAGA AGCTTCAAGAACTAGCTACTCAGCTAACTGATCTATGGAATCTGATGGATACTTCTGATGAGGAAAGGGA GCTTTTTGACCATGTTACCTCTAACATTTCAGCTTCAGTTCATGAAGTGACTGCCTCTGGTGCTCTCGCA CTTGATCTTATCGAACAGGCTGAGGTGGAAGTGGATAGGCTTGATCAACTGAAATCTAGCAGAATGAAGG AAATTGCATTCAAAAAGCAATCAGAGCTTGAGGAGATATATGCTCGTGCTCACATAGAAATAAAACCAGA GGTTGTTCGTGAGAGGATCATGTCGTTGATTGATGCTGGGAATACTGAACCTACTGAGTTACTGGCTGAC ATGGATAGCCAGATCGCAAAGGCCAAGGAGGAAGCCTTTAGTAGAAAAGAAATTTTAGACCGTGTAGAGA AATGGATGTCAGCTTGTGAGGAAGAGAGCTGGCTCGAAGATTACAATCGAGATCAGAACAGGTATAGTGC AAGCCGAGGTGCGCATTTGAATCTCAAGAGAGCTGAGAAAGCTCGGATTCTGGTCAGCAAGATTACTGCT ATGGTTGACACGTTGATTGCTAAAACCCGGGCATGGGAAGAGGAGAACAGTATGTCCTTTGAGTACGATG GAGTTCCTTTACTCGCCATGCTAGATGAATACACTATGCTTAGGCAAGAACGAGAAGATGAGAAGCGGAG GCTGAAAGAACAGAAGAAGCAGCAAGAACAGCCACACACAGATCAAGAATCCGCCTTTGGGTCTAAACCA AGCCCTGCAAGACCCGTTAGTGCCAAGAAACCGGTGGGAACACGAGTCAATGGAGGAGGGCTCAACGAAA CTCCTATGAGGCGTTTGTCTATGAACTCAAACCAGAACGGAAGCAAGTCCAAGAGAGACAGTCTGAACAA GATAGCTTCACCTTCGAACATTGTAGCCAACACAAAAGATGATGCTGCATCTCCAGTCTCTCGTGCTGAC CCAGTGATGGCTTCACCGTGACAATATCAAAAGAAAGAGAGTTATAATGCTTACTTACACTTTTGTTAGT TGCTAATAGTGCTGTTTAAGAAACAAACACTTAGAAACAAAGACAATGAGAGTAGAATGTTACTTAGATG TTTCTGTTTCTGTATTCATTCACTCAAATTTGTTTGGTACTTGTTAGCATGACTAATGTGAAGTGACCGG TTTAGTGAATGAAAGTCAAGGTTCTAATTGGAATTACGAAATAATATTTGGGC

Predicted gRNA sequences with best possible CRISPR scores:

CRISPRscan score Locus Oligo 74 64 68 67 62 72 59 82 83 66 yourseq:72.95 (-) yourseq:72.94.) yourseq 309-332 () yourseq:314-337.) yourseq:315-338 (-) yourseq:461-484 (-) yourseq:1398-1421 (-) yourseq:1733-1756 (-) yourseq:1733-1755 (-) yourseq:1783-1806 () taatacgactcactataGGGTTGGGAAATGGGAAAAGgttttagagctagaa taatacgactcactataGGTTGGGAAATGGGAAAAGgtettagagctagaa taatacgactcactataGGTGCAGGAAATCTGGGATGgrattagagctagaa taatacgactcactataGGGAAATCTGGGATGAGGTTgttttagagctagaa taatacgactcactataGGAAATCTGGGATGAGGTTGgttttagagctagaa taatacgactcactataGGGATTGGAAAGTTCAACAGgttttagagctagaa taatacgactcactataGGAATGGATGTCAGCTTGTGgttttagagctagaa taatacgactcactataGGGGCTTGGTTTAGACCCAAgtettagagctagaa taatacgactcactataGGGCTTGGTTTAGACCCAAgttttagagctagaa taatacgactcactataGGGGGAACACGAGTCAATGGgttttagagctagaa

Primer for gRNA construct predicted using CRISPRScan tool (Cas9-NGG)

gRNA selected with best score 74.

Forward primer: TAATACGACTCACTATAGGGTTGGGA [Tm 58oC]

Reverse primer: TTCTAGCTCTAAAACCTTTTCCCATTT [Tm 56oC]