Background

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a collection of DNA sequences found in bacteria that contain snippets of viral DNA from previous exposures (Marraffini and Sontheimer, 2010). The snippets are referred to as spacer DNA, and they are flanked by short, repetitive palindromic sequences. Bacteria use these stored spacer sequences as a template to express RNA to recognize and attack specific viruses if they are exposed again. When combined with CRISPR-associated (Cas) proteins, CRISPR-Cas systems can recognize and cut foreign DNA or RNA, destroying the virus and protecting the host from repeated infections (Barrangou, 2013).

A specific CRISPR system, the type II CRISPR-Cas9 from Streptococcus pyogenes, has been modified into a simpler system for use in genomic editing. With this system, researchers are able to design specific single-guide RNA (sgRNA) sequences that are complementary to a 20 bp sequence of a gene of interest that has an upstream protospacer adjacent motif (PAM; ‘NGG’) (Jinek et al., 2012). When the designed sgRNA complexes with the Cas9 protein, the assembled ribonucleoprotein binds to and introduces a double strand break (DSB) in the target DNA sequence. In genome editing applications, this DSB is then repaired by a cell’s native repair mechanisms. In the absence of an introduced repair template, the nonhomologous end-joining DNA repair pathway is normally used to repair the break in most eukaryotes (Moore and Haber, 1996). Repair via nonhomologous end-joining frequently results in an indel mutation that causes a frameshift mutation and disrupts the gene’s function. The simple programmability of the sgRNA sequences allows for unprecedented precision in genomic edits. In addition, the portability of the CRISPR-Cas9 system has allowed precise genome editing and other applications in organisms where it was previously tedious or impossible (Mali et al., 2013; Wang et al., 2013; Lobs et al., 2017b; Schwartz et al., 2017b and 2017c). The efficiency of the CRISPR-Cas9 system has been shown to correlate with sgRNA expression (Hsu et al., 2013; Ryan et al., 2014; Yuen et al., 2017). Because of this, a range of strategies for sgRNA expression have been developed. RNA polymerase II promoters, which primarily serve to drive expression of mRNA, have been used because they are widely studied and offer a high degree of control over expression (Deaner et al., 2017). More commonly for CRISPR systems, RNA polymerase III (Pol III) promoters have been used to drive sgRNA expression. Pol III promoters natively drive expression of smaller RNAs, most notably tRNAs, and yield higher transcript levels (Schwartz et al., 2016). To increase functional sgRNA expression levels even higher, Pol III promoters concatenated with tRNAs have been used (Xie et al., 2015; Schwartz et al., 2016; Lobs et al., 2017a). Implementation of synthetic hybrid Pol III promoter systems can improve CRISPR-Cas9 mediated genome editing for efficient gene disruption.