Construction of Cas13b(d), gRNA expression plasmids
The origin plasmids TC775, TC762, TC918, TC933 were purchased from
addgene
(https://www.addgene.org). For
Cas13b gRNA expression plasmid construction, SphI and SapI were used for
digestion of TC775 (PCMV::Cas13b-P2A-GFP), the resulted large fragment
was recovered by electrophoresis. U6 promoter and DR sequence of crRNA
in TC762 (PU6::PspCas13bDR) were amplified by PCR (forward primer:
5’-GAAGACAATAGCAGGcatgcGACTGCAGAGGtccgacgcc-3’, reverse primer: 5’-
AGTGAGCGAGGAAGCGGAAGAGCGATCCAAAAAAAgttgtaat-3’) according to the
condition: 2 min at 98°C, 35 cycles of 10s at 98°C, 15s at 56°C, and 20s
at 72°C, and a final step at 72 °C for 10 min. The PCR product purified
using Universal DNA Purification Kit (Tiangen) was cloned into the large
fragment with In-Fusion® HD Cloning Kit (TARAKA) to obtain the plasmid
of PCMV::Cas13b-P2A-GFP-PU6::PspCas13bDR. For the construction of Cas13d
gRNA expression plasmid, TC918 (PCAG::Cas13d-P2A-GFP) was linearized
using NotI and cloned with PCR products from TC933 (PU6:: CasRfx crRNA).
The primer sequences of PCR were as follows: (forward primer: 5’-
GGCGGGCATGCGGCCtccgacgccgccatctctag-3’, reverse primer:
GTAAGTTATGCGGCCGAGTCAGTGAGCGAGGAAGC-3’). The plasmid obtained was
PCAG::Cas13d-P2A-GFP-PU6::CasRfx-crRNA.
The guide RNAs (gRNAs) against luciferase were designed in three
batches. The first batch was based on CRISPR-Cas system targeting data
(http://chopchop.cbu.uib.no).
The second batch was based on Primer3
(https://primer3.ut.ee) to select
sequences with high specificity. The third batch of gRNAs were selected
randomly. The DNA fragments for coding selected gRNAs were synthesized
and cloned into the plasmids obtained above by BsmBI restrict enzyme
(NEB). The resulted plasmid was designed as Cas13b-gRNA or Cas13d-gRNA
respectively. All gRNA sequences were listed in Table. S1. All plasmids
were prepared using Endotoxin-free plasmid extraction kit (Tiangen,
China).