gRNA_Cloning Vector

CRISPR Human - Repression HPV-16 E6

Experiment
CRISPR Human - Repression HPV-16 E6
Product
gRNA_Cloning Vector from Addgene
Manufacturer
Addgene

Protocol tips

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

Plasmids
The hCas9 expression vector was a gift from Xingxu Huang (Addgene plasmid # 44,758) [18], and gRNA cloning vectors was a gift from George Church (Addgene plasmid # 41,824) [19]. The plasmids were prepared by using the Qiagen Endofree Plasmid Kit (Qiagen; Hilden, Germany).

Construction of gRNA Expression Plasmids
gRNA expression plasmids were constructed according to manufacturer's protocol [10]. Briefly, to prepare a 100-bp dsDNA insert fragment containing the target sequence (20 bp) and a protospacer-adjacent motif (PAM) sequence, we used a set of oligonucleotides and generated the fragment using T4 PNK (NEB; Ipswich, MA, USA). The dsDNA fragments were purified and inserted into the BbsI site of a gRNA cloning vector with T4 DNA ligase (NEB; Ipswich, MA, USA). Detailed BLAST searching of human and murine genomes was carried out to identify potential off target binding of HPV gRNAs. Two sets of oligonucleotides were designed. All oligonucleotides were synthesized and purified by Sangon Biotech Co. (Shanghai, China). The sgRNAs specific to HPV16 E6 gene were 5′-CACCGCAACAGTTACTGCGACGTG-3′, and 5′-AAACCACGTCGCAG TAACTGTTGC-3′. The sgRNAs specific to HPV16 E7 gene were 5′-CACCGACACGTAGACATTCGTACTT-3′, and 5′-AAACAAGTACGAAT GTCTACGTGT-3′ [10].

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Discussion

Discussion

1 year ago

Author: Milena Alexeyeva Russian Federation

DNA insert using CRISPR

I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?

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Papers

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Manufacturer protocol

Download the product protocol from Addgene for gRNA_Cloning Vector below.

Download PDF Download manufacturer protocol

Videos

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