hCas9

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Protocol tips
There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

CRISPR knockouts
Candidate target sequences for CRISPR were designed using ZiFiT Targeter Version 4.2 (http://zifit.partners.org/ZiFiT/). The sequences of guide RNA were placed in pENTR221-U6-gRNA by inverse PCR, as previously described29. hCas9 was purchased from addgene (Plasmid #41815). hCas9 was PCR amplified and transferred to pENTE221 by standard BP Clonase reaction (Invitrogen), following manufacturers protocol. hCas9 was then transferred to PB-TRE-DEST-EF1A-rtTA-RES-Puro29 by standard LR Clonase reaction (Invitrogen), following manufacturers protocol, to generate PB-TRE-Cas9-EF1A-rtTA-IRES-Puro. The Gateway DEST cassette was then PCR amplified with NheI site engineered into the primers and cloned into a unique NheI site upstream of the TRE promoter to generate PB-DEST-TRE-Cas9-EF1A-rtTA-IRES-Puro. The guide RNAs were then transferred to PB-DEST-TRE-Cas9-EF1A-rtTA-IRES-Puro via standard LR Clonase reaction (Invitrogen), following manufacturers protocol. Two micrograms of each PB-U6-gRNA-TRE-Cas9-EF1A-rtTA-IRES-Puro and Super piggyBac transposase (System Biosciences) were transfected to B117P by NEON® Transfection System (Life Technologies Corporation, Carlsbad, CA) using 1,400 volts and 20 milliseconds for 2 pulses. Two days later transfected cells were selected with 1.5 μg/ml puromycin and 1.0 μg/ml of doxycycline for more than 3 weeks to generate stable knockout cell lines. DNA was collected using standard Phenol:Chloroform extraction. A CEL-I assay was performed (Supplementary Figure S5) and the gene modification ratio calculated, as previously described30.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Mouse - Deletion Dck using hCas9 from Addgene.

Manufacturer protocol

Download the product protocol from Addgene for hCas9 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Mouse - Deletion Dck using hCas9 from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.