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URL: https://dx.doi.org/DOI:10.17504/protocols.io.brbnm2me

Authors: Olin Silander

Summary: For CRISPR-cas9 protocols including BAC-PULCE, T7 polymerase is used to transcribe the crRNA and tracrRNA to make dgRNA for cas9. The two components of the dual guides are the crRNA (containing your variable 20 nt target plus a 22 nt constant region) and the tracrRNA (a 72 nt constant region). We used a combination of these protocols: https://www.protocols.io/view/in-vitro-transcription-for-dgrna-3bpgimn /dx.doi.org/10.17504/protocols.io.3bpgimnhttps://international.neb.com/protocols/2013/04/02/standard-rna-synthesis-e2050Nanopore protocol Cas-mediated PCR-free enrichment - please refer to this protocol in particular for further important detail around sequencing the DNA library. Using modified “In Vitro Transcription for dgRNA V.2” (Lyden et al 2019):In this protocol, we are designing crRNAs against which Cas9 will target. Using this sequence, we will add a T7 RNA polymerase binding site at the 5’ end, a 3’ tracrRNA binding site, and use the reverse complement of this to order our DNA oligos (below more details):Designing crRNAs using CHOPCHOPv3 (Labun et al 2019), insert your gene target and CHOPCHOP will find a sequence of 20nt’s that will end in an  ‘NGG-3’5’----NNNNNNNNNNNNNNNNNNNNNGG----3’where 20Ns are your target site. Cas9 cuts between the 17th and 18th N of your target.The sequence of each crRNA should be as follows, with the Ns replaced by your 20 nt target:TAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTATGCTGTTTTGThe underline portion is the T7 RNA polymerase binding site. The addition at the 3’ end represents the region to which an 18mer will bind, in order to allow T7 a double stranded binding site.18mer T7:TAATACGACTCACTATAG

Affiliations: Massey University

Version: 1

Publication Date: 2021

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