Does CRISPR work in all species? Take a look at those amazing CRISPR Plasmids and you’ll know the answer

As an open biological material sharing platform, MolecularCloud has received 100+ plasmids this year from Cloud Scientists for genome editing in E. coil, Yeast, Mammalian, Rice, Plant, Bacillus subtilis, etc. Here, I want to share some of those popular plasmids with you.

CRISPR Plasmids for E. coil

Plasmid Name: pQCascade, pDonor-GDH, etc. 

Depositor: Sheng Yang 

Description: Dr. Yang’s team demonstrated that multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) can be achieved by designing a crRNA to target multicopy loci or a crRNA array to target multiple loci in the Escherichia coli genome. Within 5 days without selection pressure, E. coli strains carrying cargos with successively increasing copy numbers (up to 10) were obtained. Recombinant MUCICAT E. coli containing genomic multicopy glucose dehydrogenase expression cassettes showed 2.6-fold increased expression of this important industrial enzyme compared to E. coli harboring the conventional protein-expressing plasmid pET24a. Successful extension of MUCICAT to Tatumella citrea further demonstrated that MUCICAT may be generally applied to many bacterial species.

Plasmid Name: pAPOBEC_nCas9_Ung etc.

Depositor: Changhao Bi 

Description: Dr. Bi’s team presents BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process.

CRISPR Plasmids for Bacillus subtilis

Plasmid Name: pHT-XCR6, pcrF11 etc.

Depositor: Long Liu

Description: Dr. Liu’s team built a powerful tool called CRISPR/Cpf1 assisted multiple-genes editing and regulation system for B. subtilis, which was constructed for engineering Bacillus subtilis. And synthetic oligos mediated assembly of CRISPR RNA (crRNA) array method was created to build crRNA array. This system can achieve the double genes in-frame knocking out, multiple point mutations (up to six), or single gene insertion at a time with 100% efficiency.

CRISPR Plasmids for Mammalian

Plasmid Name: pCmv-BEACON1, pCmv-BEACON2, etc.

Depositor: Jia Chen 

Description: Dr. Jia Chen’s team demonstrated that the original version of catalytically dead Cas12a (dCas12a)-conjugated BEs induce a basal level of DNA breaks and minimally activate DDR proteins, including H2AX, ATM, ATR, and p53. They went on further to develop a dCas12a-based BEACON (base editing induced by human APOBEC3A and Cas12a without DNA break) system through fusing dCas12a with engineered human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A), and successfully applied it for both in vitro and in vivo editing. The BEACON system achieves efficient and specific base editing without generating DNA breaks or triggering DDR cascades, which is essential for its broad applications in mammalian cells and in clinics.