gRNA_Cloning Vector

CRISPR Human - Repression HPV-16 E7

CRISPR Human - Repression HPV-16 E7
gRNA_Cloning Vector from Addgene

Protocol tips

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

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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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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Manufacturer protocol

Download the product protocol from Addgene for gRNA_Cloning Vector below.

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