Part:BBa_K4667000

Rna-mediated endonuclease can specifically cut target double-stranded DNA in the presence of PAM in

Cas12a (Cpf1) is a CRISPR-related nuclease

The use of Cas12a (1) Cas12a requires only a single crRNA and is smaller and easier to deliver into cells; (2) Cas12a produces sticky ends after cutting, which is more conducive to accurate genome editing; (3)The cutting site of Cas12a is far away from its recognition site, which provides the possibility of continuous editing; (4)the PAM sequence recognized by Cas12a is different from that of Cas9, thus providing a choice of different editing sites; For different Cas12a proteins, the required PAM sites are different, in which some Cas12a proteins recognize TTN sites and some Cas12a proteins recognize TTTN sites.

Sequence and Features BBa_K4667000 SequenceAndFeatures Procedure for plasmid transformation a) Take one TOP10 receptor cell containing 50μL ~200L (stored at 80C) and thaw it on ice for 20-30 minutes: b) Take 2~3μl (dissolve the freeze-dried 4ug plasmid into 100ng/ul with sterile water, refer to the instructions in Answer 4 above for details) and transfer it into the thawed receptive cells (gently pick it with your fingers); c) Place on ice for 10-15 minutes: d) Place 2/3 of it in a 42C water bath for 120 seconds for heat hammering: e) Immediately remove from the water bath and return to the ice for 2-5 minutes: f) Add 800! ILB medium and grow in 37C oscillating incubator (200rpm) for 40 minutes; g) convert some or all of it to contain 100! g/m1 antibiotic on an LB AGAR plate; h) plate inverted, 37"C overnight culture.

Engineering Success

We have designed the pET-Cas12a expression plasmid. To construct our plasmid, we let the company synthesise DNA fragments by inserting FnCas12 into the pET28a plasmid vector. The constructed plasmids were contained in E. coli strains, we streak inoculated them on LB liquid medium plates containing corresponding antibiotics and incubated them at 37℃ overnight

A. E. coli BL21 containing the pET28a plasmid. B. E. coli BL21 containing the pET28a plasmid. C. pET28a plasmid after single colony culture. D. pET28a plasmid after single colony culture. The results showed that the plasmid could grow on LB medium containing kanamycin resistance. Sequence and Features BBa_K4667000 SequenceAndFeatures We performed Cas protein purification experiments and obtained sufficient concentrations of Cas12a protein.

A. Incubation of Cas12a containing the pET28a plasmid.
B. SDS-PAGE electrophoresis gels of Cas12a protein after IPTG induction at different concentrations.

Standard linear regression line of BCA method used to calculate protein concentration. Absorbance and calculated protein concentration of Cas12a. Through the curve values, we measured the absorbance of Cas12a as 0.2276 (L/(g.cm)) and the protein concentration (ug/ml) as 10.7549295, this result indicates that we obtained a sufficient concentration of Cas12a protein.

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