pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA
Plasmid 61591

SaCas9 User Manual

PAM sequence

The SaCas9 PAM sequence for optimal on-target cutting isNNGRRT. Nevertheless, targets with PAMs of the formNNGRR(N) also have appreciable cutting and should beconsidered in evaluating off target activity.Optimal guide lengthThe optimal guide length for SaCas9 is 21- or 22-nt. If theguide does not begin with a “G,” it is advisable to add a G tothe 5’ to enhance transcription from the U6 promoter.Cloning of GuidesCloning the guide into the vectorcan be done using the Type IIsrestriction site BsaI, whichallows scarless insertion of the21- or 22-bp guide.Guide sequences can beordered as standard oligos with5’-CACC and 5’-AAACoverhangs and “G” addition asillustrated. For details with PNKtreatment,annealing of oligos,and digestion of vector, pleasesee:http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-ReagentDescription-Rev20140509.pdf.Note that the Type IIs sites for SpCas9 plasmids are BbsI and those for SaCas9 are BsaI, withcloning instructions otherwise identical.Alternatively, the entire U6::sgRNA region can be replaced by restriction site-flanked PCR ampliconof the appropriate guide. PCR amplicons allow high-throughput screening of guides when cotransfectedwith SaCas9, and the highest efficiency U6::sgRNA can be cloned into a single vectorwith SaCas9. See Ran et al., Nature Protocols 2013 for details:(http://www.nature.com/nprot/journal/v8/n11/abs/nprot.2013.143.html).Guide testing workflowGuides can be tested in a cell line from the relevant species. In general, we have found that guidesthat work in cell lines will also work when delivered to in vivo tissue, though efficiencies may differ.Additionally, AAV purity and accurate titration is essential to getting efficient cutting in vivo.

Sequence序列：

>pX601

cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccgg

cctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggcctcta

gactcgaggcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcata

gcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgaccc

ccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaa

tgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcccc

ctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcc

tacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaat

gggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgt

tttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcgg

taggcgtgtacggtgggaggtctatataagcagagctctctggctaactaccggtgccaccATGGCCCCA

AAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCAAGCGGAACTACATCCTGGGCCTGGACA

TCGGCATCACCAGCGTGGGCTACGGCATCATCGACTACGAGACACGGGACGTGATCGATGCCGGCGTGCG

GCTGTTCAAAGAGGCCAACGTGGAAAACAACGAGGGCAGGCGGAGCAAGAGAGGCGCCAGAAGGCTGAAG

CGGCGGAGGCGGCATAGAATCCAGAGAGTGAAGAAGCTGCTGTTCGACTACAACCTGCTGACCGACCACA

GCGAGCTGAGCGGCATCAACCCCTACGAGGCCAGAGTGAAGGGCCTGAGCCAGAAGCTGAGCGAGGAAGA

GTTCTCTGCCGCCCTGCTGCACCTGGCCAAGAGAAGAGGCGTGCACAACGTGAACGAGGTGGAAGAGGAC

ACCGGCAACGAGCTGTCCACCAAAGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAATACGTGG

CCGAACTGCAGCTGGAACGGCTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAAGACCAG

CGACTACGTGAAAGAAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGACCAGAGCTTC

ATCGACACCTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGACCTGGCGAGGGCAGCCCCT

TCGGCTGGAAGGACATCAAAGAATGGTACGAGATGCTGATGGGCCACTGCACCTACTTCCCCGAGGAACT

GCGGAGCGTGAAGTACGCCTACAACGCCGACCTGTACAACGCCCTGAACGACCTGAACAATCTCGTGATC

ACCAGGGACGAGAACGAGAAGCTGGAATATTACGAGAAGTTCCAGATCATCGAGAACGTGTTCAAGCAGA

AGAAGAAGCCCACCCTGAAGCAGATCGCCAAAGAAATCCTCGTGAACGAAGAGGATATTAAGGGCTACAG

AGTGACCAGCACCGGCAAGCCCGAGTTCACCAACCTGAAGGTGTACCACGACATCAAGGACATTACCGCC

CGGAAAGAGATTATTGAGAACGCCGAGCTGCTGGATCAGATTGCCAAGATCCTGACCATCTACCAGAGCA

GCGAGGACATCCAGGAAGAACTGACCAATCTGAACTCCGAGCTGACCCAGGAAGAGATCGAGCAGATCTC

TAATCTGAAGGGCTATACCGGCACCCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGCTG

TGGCACACCAACGACAACCAGATCGCTATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGTGGACCTGT

CCCAGCAGAAAGAGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTCGTGAAGAGAAGCTT

CATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAACGACATCATTATCGAG

CTGGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAACGAGATGCAGAAGCGGAACCGGCAGA

CCAACGAGCGGATCGAGGAAATCATCCGGACCACCGGCAAAGAGAACGCCAAGTACCTGATCGAGAAGAT

CAAGCTGCACGACATGCAGGAAGGCAAGTGCCTGTACAGCCTGGAAGCCATCCCTCTGGAAGATCTGCTG

AACAACCCCTTCAACTATGAGGTGGACCACATCATCCCCAGAAGCGTGTCCTTCGACAACAGCTTCAACA

ACAAGGTGCTCGTGAAGCAGGAAGAAAACAGCAAGAAGGGCAACCGGACCCCATTCCAGTACCTGAGCAG

CAGCGACAGCAAGATCAGCTACGAAACCTTCAAGAAGCACATCCTGAATCTGGCCAAGGGCAAGGGCAGA

ATCAGCAAGACCAAGAAAGAGTATCTGCTGGAAGAACGGGACATCAACAGGTTCTCCGTGCAGAAAGACT

TCATCAACCGGAACCTGGTGGATACCAGATACGCCACCAGAGGCCTGATGAACCTGCTGCGGAGCTACTT

CAGAGTGAACAACCTGGACGTGAAAGTGAAGTCCATCAATGGCGGCTTCACCAGCTTTCTGCGGCGGAAG

TGGAAGTTTAAGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCGAGGACGCCCTGATCATTGCCAACG

CCGATTTCATCTTCAAAGAGTGGAAGAAACTGGACAAGGCCAAAAAAGTGATGGAAAACCAGATGTTCGA

GGAAAAGCAGGCCGAGAGCATGCCCGAGATCGAAACCGAGCAGGAGTACAAAGAGATCTTCATCACCCCC

CACCAGATCAAGCACATTAAGGACTTCAAGGACTACAAGTACAGCCACCGGGTGGACAAGAAGCCTAATA

GAGAGCTGATTAACGACACCCTGTACTCCACCCGGAAGGACGACAAGGGCAACACCCTGATCGTGAACAA

TCTGAACGGCCTGTACGACAAGGACAATGACAAGCTGAAAAAGCTGATCAACAAGAGCCCCGAAAAGCTG

CTGATGTACCACCACGACCCCCAGACCTACCAGAAACTGAAGCTGATTATGGAACAGTACGGCGACGAGA

AGAATCCCCTGTACAAGTACTACGAGGAAACCGGGAACTACCTGACCAAGTACTCCAAAAAGGACAACGG

CCCCGTGATCAAGAAGATTAAGTATTACGGCAACAAACTGAACGCCCATCTGGACATCACCGACGACTAC

CCCAACAGCAGAAACAAGGTCGTGAAGCTGTCCCTGAAGCCCTACAGATTCGACGTGTACCTGGACAATG

GCGTGTACAAGTTCGTGACCGTGAAGAATCTGGATGTGATCAAAAAAGAAAACTACTACGAAGTGAATAG

CAAGTGCTATGAGGAAGCTAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGTTTATCGCCTCCTTCTAC

AACAACGATCTGATCAAGATCAACGGCGAGCTGTATAGAGTGATCGGCGTGAACAACGACCTGCTGAACC

GGATCGAAGTGAACATGATCGACATCACCTACCGCGAGTACCTGGAAAACATGAACGACAAGAGGCCCCC

CAGGATCATTAAGACAATCGCCTCCAAGACCCAGAGCATTAAGAAGTACAGCACAGACATTCTGGGCAAC

CTGTATGAAGTGAAATCTAAGAAGCACCCTCAGATCATCAAAAAGGGCAAAAGGCCGGCGGCCACGAAAA

AGGCCGGCCAGGCAAAAAAGAAAAAGggatcctacccatacgatgttccagattacgcttacccatacga

tgttccagattacgcttacccatacgatgttccagattacgcttaagaattcctagagctcgctgatcag

cctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctgga

aggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcat

tctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctg

gggaggtacctgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagaga

gataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataa

tttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaa

agtatttcgatttcttggctttatatatcttGTGGAAAGGACGAAACACCgGAGACCacggcaGGTCTCa

gttttagtactctggaaacagaatctactaaaacaaggcaaaatgccgtgtttatctcgtcaacttgttg

gcgagatttttgcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgc

tcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcg

agcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacac

cgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttac

gcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctc

gccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctt

tacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagac

ggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaaca

ctcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaa

atgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcac

tctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcg

ccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgt

gtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttata

ggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacc

cctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgc

ttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgc

ggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttg

ggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaag

aacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgg

gcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaa

aagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactg

cggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggggga

tcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacacc

acgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttccc

ggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggc

tggctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactgggg

ccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa

atagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcata

tatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataat

ctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaag

gatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc

ggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcag

ataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgccta

catacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggtt

ggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagccc

agcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttc

ccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagct

tccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattt

ttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctgg

ccttttgctggccttttgctcacatgt