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Genome editing in the choanoflagellate Salpingoeca rosetta v2
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This protocol details the preparation and execution of CRISPR/Cas9 genome editing in S. rosetta. The protocol builds on a method to transfect macromolecules into S. rosetta for delivering a purified Cas9 ribonucleoprotein from Streptomyces pyogenes (SpCas9 RNP) into S. rosetta. Upon cleaving the S. rosetta genome at locations specified by the guide RNA (gRNA) of the SpCas9 RNP, S. rosetta can use DNA oligonucleotides as templates to repair the double-stranded break. Those repair templates can encode foreign sequences and mutations for editing the S. rosetta genome, so long as DNA oligonucleotides have >30 bases of sequence that is homologous to both sides of the Cas9 cleavage site.
Title: Genome editing in the choanoflagellate Salpingoeca rosetta v2
Description:
This protocol details the preparation and execution of CRISPR/Cas9 genome editing in S.
rosetta.
The protocol builds on a method to transfect macromolecules into S.
rosetta for delivering a purified Cas9 ribonucleoprotein from Streptomyces pyogenes (SpCas9 RNP) into S.
rosetta.
Upon cleaving the S.
rosetta genome at locations specified by the guide RNA (gRNA) of the SpCas9 RNP, S.
rosetta can use DNA oligonucleotides as templates to repair the double-stranded break.
Those repair templates can encode foreign sequences and mutations for editing the S.
rosetta genome, so long as DNA oligonucleotides have >30 bases of sequence that is homologous to both sides of the Cas9 cleavage site.
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