A Novel System for the Rapid Generation of Precise DNA Deletions

To generate DNA deletions, a tandem array of class IIS restriction enzyme recognition sites was cloned into a plasmid. The recognition sites were arranged so that each enzyme cleaves at a different site within an adjacent target sequence. Digestion with both enzymes followed by end repair and ligation resulted in the deletion of DNA between the two sites of cleavage. Because both recognition sites are preserved following deletion, it was found that sequential deletions could be generated using cycles of restriction enzyme digestion, end repair and ligation. Therefore, this system represents a valuable tool in the definition of functional DNA sequences. The analysis of transcriptional regulatory elements within a region of DNA typically involves deletion generation using the exonuclease III/mung bean nuclease system (1). Initially, large deletions are created and regulatory elements are mapped in vivo using a reporter gene system. Further deletions are often required to precisely define regulatory elements and this usually involves the generation of large numbers of deletions. While the exonuclease III/mung bean nuclease deletion system is a routine procedure, the irregular rate and sequence dependency of exonucleolytic digestion [personal observations; (2)] render the generation of the large numbers of random deletions time consuming and expensive. Another common way to generate defined DNA deletions is using the polymerase chain reaction (PCR). However, PCR-based strategies for the generation of deletions have several drawbacks. Chiefly, primer Tm and secondary structure considerations constrain the sizes and locations of deletions. Additionally, if multiple deletions are to be generated, then the expense of the multiple primers required often limits the number of deletions possible, thereby further reducing the resolution of regulatory element mapping. Furthermore, following amplification, each of the deletions are cloned thus increasing the time required to map elements. Here we describe a system that allows the rapid, sequential generation of small, predictable deletions of DNA sequences. The deletions generated using this system are not dependent on the sequence to be deleted or flanking sequences, and therefore do not suffer from the drawbacks of the PCR or exonuclease III/mung bean nuclease based systems. The system relies on the property of class IIS restriction enzymes of cleaving DNA at a specific site that is distant from the recognition sequence. We have exploited this property to generate DNA deletions by the insertion of a cassette into the commonly used cloning vector pBluescript II SK+ (Stratagene), to produce the vector pNBL. The cassette contains a tandem arrangement of the recognition sequences of two of these class IIS enzymes, BsgI and BsmBI (Fig. 1a). Each enzyme cleaves at a different distance from the recognition site, therefore, the sequence between the sites of cleavage is deleted upon digestion with both of the enzymes (Fig. 1b). Therefore, the DNA fragment to be deleted is (sub-)cloned 34 to the deletion cassette. As depicted in Figure 1, this tandem array is organized such that the recognition sequence of both enzymes remains intact following the procedure, allowing further rounds of deletion. Various sizes of deletion can be produced depending on the method of repair of the resulting staggered ends prior to religation