Multiplex CRISPR/Cas9 Assembly System Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.	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.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.
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

Plasmid construction for human cell experiments
The CRISPR/Cas9 vector expressing Cas9 nuclease and an sgRNA targeting EGFP reporter vector was constructed using pX330 vector (Addgene Plasmid #42230) according to the previously described protocol [46]. The all-in-one CRISPR/Cas9 vector expressing Cas9 nuclease and two sgRNAs targeting the FBL or hACTB gene and the donor vector was constructed using the Multiplex CRISPR/Cas9 Assembly System Kit (Addgene Kit #1000000055) as described previously [30]. The oligonucleotide sequences for sgRNA templates were listed in Additional file 1: Table S4.

The overexpression vectors were constructed using an RT-PCR and In-Fusion HD Cloning Kit. Briefly, the full coding sequences for the 14 genes listed in Fig. 2d were amplified using an RT-PCR from total RNAs extracted from HEK293T cells. The amplified cDNAs were cloned into ptCMV-136/63-VR-NG vector (Addgene Plasmid #50700) [47], replacing the transcription activator-like effector nuclease-coding sequence with each cDNA. The sequences of primers used to construct the overexpression vectors were listed in Additional file 1: Table S4.

The EGFP reporter vector for monitoring MMEJ frequency was constructed using a PCR and In-Fusion HD Cloning Kit (Takara). PITCh(gRNA-s1)-FBL and PITCh(gRNA-s1)-hACTB donor plasmids were constructed using a PCR and TA-cloning with DynaExpress TA PCR Cloning Kit (pTAC-2) (BioDynamics Laboratory Inc.) or TArget Clone -Plus- (Toyobo). The full plasmid sequences of EGFP reporter vector, PITCh(gRNA-s1)-FBL donor vector, and PITCh(gRNA-s1)-hACTB donor vector are shown in Additional file 1: Figure S17.



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