gRNA_Cloning Vector

Protocol tips

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	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

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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Papers

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Manufacturer protocol

Download the product protocol from Addgene for gRNA_Cloning Vector below.

Download manufacturer protocol

Videos

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