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 NamepHT-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. 

Plasmid Name: pCMV-dCpf1-BE0 etc.

Depositor: Jia Chen

Description: The targeting range of CRISPR–Cas9 base editors (BEs) is limited by their G/C-rich protospacer-adjacent motif (PAM) sequences. To overcome this limitation, Dr. Jia Chen’s team developed a CRISPR–Cpf1-based BE by fusing the rat cytosine deaminase APOBEC1 to a catalytically inactive version of Lachnospiraceae bacterium Cpf1. The base editor recognizes a T-rich PAM sequence and catalyzes C-to-T conversion in human cells, while inducing low levels of indels, non-C-to-T substitutions and off-target editing.

CRISPR Plasmids for Plant

Plasmid Name: pVS1-VIR2, pRiA4-VIR,etc.

Depositor: Qijun Chen

Description: Dr. Chen’s team demonstrated that morphogenic regulator (MR) vectors greatly enhance maize (Zea mays) transformation. They then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, they developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. The ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.

Plasmid Name: pHUC411-gcccc,pHUC411NG-gcccc, etc.

Depositor: Pengcheng Wei

Description: Dr. Pengcheng Wei found that SpCas9-NG not only targeted the genome but also efficiently self-targeted the single-guide RNA sequence in transfer DNA in transgenic plants, potentially increasing off-target risk by generating new single-guide RNAs. They further showed that the self-target effect of SpCas9-NG could be greatly alleviated by using a modified single-guide RNA scaffold starting with a GCCCC sequence. 

Plasmid Name: pPUN411-ABEH

Depositor: Pengcheng Wei

Description: Dr. Wei’s team provided an efficient and easy-to-use ABE system for plantadenine base editing. More importantly, the study offers a general strategy to optimize the efficiency of stable transformationbased plant genome editing. 

Do you have any questions about above plasmids or CRISPR genome editing technology?

What problems have you encountered  in CRISPR experiments? 

Leave your thoughts now in the comment section below where they will be answered soon by our scientists in various fields involving E. coil, Yeast, Mammalian, Rice, Bacillus subtilis, etc. Additionally, the best comments will also give your a chance to win a canvas bag, hoodie or $20 Amazon/50 JD gift card

Tips: Go to the depositor's personal page to find more information of all plasmids.


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