Gibson assembly snapgene drivers#
Gibson assembly snapgene series#

To overcome this technical chasm, the Jacks lab at MIT used a Gibson Assembly-based modular assembly platform (GMAP a technique used to generate a series of modular vectors for bacterial and mammalian gene expression) to construct a CRISPR-mediated system for testing candidate tumor suppressor genes in mSCLC.

Gibson assembly snapgene drivers#

Д«ut untangling the role of additional tumor suppressor or oncogenic drivers of mSCLC requires the introduction of germline or conditional alleles for candidate genes into pre-existing GEMMs, a technically challenging process that can take multiple months to years. For instance, through conditional deletion of Trp53 and Rb1 in the lung epithelium, researchers have developed a robust mouse model for small cell lung cancer (mSCLC). Many GEMMs that mimic different clinical aspects of cancer have been developed, helping cancer researchers untangle the process of tumor evolution and metastasis. Finally, traditional restriction cloning of multiple fragments can be burdensome, requiring subsequent rounds of multi-day digestion and screening protocols to obtain the desired DNA construct. Restriction enzymes also have varying degrees of activity, which can lead to inefficiencies in DNA digestion. Commonly used restriction enzymes cut inverted repeat palindromic hexanucleotide sequences and dependence on these restriction sites has limitations: They must be incorporated into DNA fragments before cloning which can introduce undesirable sequences (also called вЂњscarsвЂќ) into gene products. Reliance on conventional cloning workflows-which use restriction enzymes-can present additional hurdles for molecular biologists looking to harness the power of CRISPR-Cas systems in their research. While this methodology presents significant advantages (i.e., circumventing expression or off-target editing complications), some experimental questions may require the use of reporter or selection markers, which necessitates the use of plasmid or virus-based systems, constructed using recombinant DNA technology. Briefly, RNPs are assembled in vitro and transfected or electroporated into the desired cells. One way around these difficulties is by using a purified ribonucleoprotein (RNP) complex, containing a Cas protein and sgRNA, for genome editing. Commonly, especially when implementing CRISPR-Cas expression in mammalian systems using lentiviruses, researchers can face problems with unstable inserts that undergo homologous recombination during the cloning process.

These requirements can act as a major bottleneck to implementing CRISPR protocols in several model systems. Accomplishing this requires a modular approach to constructing recombinant DNA, a robust cloning workflow, and rapid access to custom PCR primers and ready-made vectors. Wielding this technology, either for preclinical R&D or therapeutics development, requires the ability to manipulate and test various Cas proteins, sgRNAs, and other genes common in molecular biology, including fluorescent or luminescent reporters, promoters of different strengths, transcriptional terminators, and more.