Pcs2 cas9 msa

Manufactured by Addgene

The PCS2+Cas9-mSA is a gene editing tool that facilitates the delivery of Cas9 protein and single guide RNA (sgRNA) into cells. It enables efficient CRISPR-Cas9 mediated genome editing.

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Lab products found in correlation

Pt7 cas9, by OriGene (1 mentions) Mmessage mmachine t7 ultra kit, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Mmessage mmachine sp6 transcription kit, by Thermo Fisher Scientific (1 mentions) Megaclear transcription clean up kit, by Thermo Fisher Scientific (1 mentions) Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)

2 protocols using pcs2 cas9 msa

Cas9 RNA In Vitro Transcription

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Cas9 or Cas9-streptavidin plasmid (this protocol is intended for plasmids containing a T7 promoter)○

Cas9: pT7-Cas9 (available commercially from OriGene Cat. No. GE100014)

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Cas9-streptavidin: PCS2+Cas9-mSA (available from Addgene Plasmid No. 103882)○

NOTE: The SP6 promoter in this plasmid was replaced with a T7 promoter using standard cloning techniques.

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NOTE: Protocol can be adapted to use a plasmid containing an SP6 promoter.

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Restriction enzyme appropriate to linearize Cas9 plasmid (see note 1c)

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Phenol/Chloroform

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3 M NaOAc, pH 5.3

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100% ethanol

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mMessage mMachine T7 Ultra kit (Thermo Fisher Cat. No. AM1345)•

NOTE: Use SP6 in vitro transcription kit if using a plasmid containing an SP6 promoter.

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RNeasy Mini Kit (Qiagen Cat. No. 74104)

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Dry ice

Pineault K.M., Novoa A., Lozovska A., Wellik D.M, & Mallo M. (2019). Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse. MethodsX, 6, 2088-2100.

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CRISPR Cas9 mRNA and sgRNA Synthesis

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Plasmid pCS2+Cas9-mSA (Addgene plasmid #103882), a kind gift from Janet Rossant [14 (link)] was linearized with Not I and used as a template for in vitro transcription with the mMESSAGE mMACHINE Sp6 Transcription kit (ThermoFisher) according to the manufacturer’s instructions. RNA was purified using the MEGAClear Transcription Clean-Up Kit (ThermoFisher) and further ethanol precipitated. sgRNA templates were prepared as described in Bassett et al. [51 (link)], with primers LA137 and LA138 (sgRNA-64), LA137 and LA2676 (sgRNA-145), LA137 and LA139 (sgRNA-190), LA137 and LA140 (sgRNA-234). sgRNAs were transcribed using the MEGAScript T7 Transcription Kit (ThermoFisher) and purified using the MEGAClear Transcription Clean-Up Kit (ThermoFisher).

Ang J.X., Nevard K., Ireland R., Purusothaman D.K., Verkuijl S.A., Shackleford L., Gonzalez E., Anderson M.A, & Alphey L. (2022). Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source. PLoS Genetics, 18(2), e1010060.

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