Leveraging Circular Polymerization and Extension Cloning (CPEC) Method for Construction of CRISPR Screening Libraries

Background

Our knowledge of the genetic and molecular aspects of cancer has advanced considerably owing to high-throughput functional genomics, which facilitates the identification of potential treatment targets or therapeutic vulnerabilities for a range of cancer types [1]. Genome-wide or focused CRISPR screening strategy has proven to be a versatile tool to unravel genes and even alterations in pathways having a major role in the survival and fitness of cancer cells, as well as therapy resistance [2,3]. Researchers can uncover details about gene functions, interactions, and regulatory networks by creating libraries containing different CRISPR constructs [4]. This understanding is crucial for deciphering complex biological systems and diseases.

Despite the availability of genome-wide or focused CRISPR libraries from commercial or repository sources, the creation of custom-built CRISPR libraries holds paramount importance for several reasons. First and foremost, de novo library construction enables the incorporation of a specific number of gRNAs, rather than being constrained by a predetermined or limited quantity. In contrast to genome-wide commercial libraries, which typically include a limited number of gRNAs per gene, custom-built libraries allow for a higher number of gRNAs per gene, thereby improving the reliability of the observed phenotypes. Moreover, to refine results and eliminate false negative candidates, secondary screens that focus on potential hits identified in primary screens are often conducted. To accomplish this, constructing a de novo library using gene sets that have already been pre-screened would become increasingly important. Second, the composition of the library can be adjusted with respect to the focus of the research. Although this study explores the CRISPR library within the context of cancer research, its application extends beyond this field. To exemplify, engineering gRNAs targeting transcription factors, metabolic genes, and immune regulation genes can be prioritized based on the specific research focus. Last but not least, a vector backbone with a Cas9 expression cassette or one that lacks Cas9 can be chosen.

In this context, researchers exploit cloning methodologies, which are classified into two primary categories: those that are sequence-dependent (Gateway cloning) and those that are sequence-independent [5], typically based on homologous recombination. Sequence-dependent strategies utilize restriction digestion-ligation methods or site-specific recombination and necessitate unique and specific sites within the insert, vector, or both. This dependence on unique sites makes these approaches less ideal for multi-fragment cloning. On the other hand, sequence-independent approaches comprise of ligation independent cloning (LIC) [6], sequence and ligation-independent cloning (SLIC) [7], Gibson assembly [8], and covalently-closed-circular synthesized (3Cs) [9]. All of these methods have pros and cons, including circular polymerization extension cloning (CPEC); however, CPEC could be exploited for the construction of CRISPR libraries whenever someone opts for a more cost-effective and streamlined approach.

Circular polymerase extension cloning (CPEC) represents a relatively cost-effective, high-efficiency cloning technique compared to the Gibson assembly and other protocols used in molecular cloning. It operates on the principle of polymerase overlap extension and is considered a robust alternative due to its omission of restriction digestion, ligation, and other procedural steps [10]. CPEC enables the transformation of overlapping DNA fragments into a double-stranded circular form via the polymerase extension mechanism, thereby facilitating the integration of the insert into the target plasmid. During the CPEC reaction, linear double-stranded inserts and vectors are first separated through increasing temperature (denaturation). Subsequently, the resulting single-stranded products anneal through their overlapping regions and use each other as templates to construct the circular plasmid. Through the CPEC method, not only a single gene but also multi-fragment assembly could be obtained. From this point of view, CPEC could have a pivotal role in the construction of CRISPR libraries (de novo). In this method, it is crucial to uniquely select the overlapping regions of the insert and vector, and the melting temperature (T_m) should be as high as possible to minimize the likelihood of vector self-ligation and concatenation.

Here, we present a robust, cost-effective, and straightforward CPEC-based protocol to engineer a custom-built gRNA library for CRISPR screening research. The knockout gRNA library, enriched for transcription factors and epigenetic regulators as well as nuclear factors, hence named EpiTransNuc, was constructed by exploiting the PCR linearized lentiGuide-Puro backbone and comprises 40820 gRNAs, comprising 10 gRNAs per gene and 100 non-targeting controls.