CRISPR–Cas9-mediated genome editing and guide RNA design

CRISPR-Cas9-mediated genome editing and guide RNA design Michael V. Wiles 0 1 Wenning Qin 0 1 Albert W. Cheng 0 1 Haoyi Wang 0 1 0 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences , Beijing , People's Republic of China 1 The Jackson Laboratory , 600 Main Street, Bar Harbor, ME 04609-1500 , USA CRISPR and CRISPR-associated (Cas) proteins, which in nature comprise the RNA-based adaptive immune system in bacteria and archaea, have emerged as particularly powerful genome editing tools owing to their unrivaled ease of use and ability to modify genomes across mammalian model systems. As such, the CRISPR-Cas9 system holds promise as a ''system of choice'' for functional mammalian genetic studies across biological disciplines. Here we briefly review this fast moving field, introduce the CRISPR-Cas9 system and its application to genome editing, with a focus on the basic considerations in designing the targeting guide RNA sequence. - Introduction Site-directed DNA endonucleases are powerful tools for genome editing. When introduced into cells, these proteins can bind to a target DNA sequence in the genome and create a DNA double-strand break (DSB), the repair of which leads to varied DNA sequence modifications. The initial efforts on developing these tools were focused on engineering homing endonucleases (Silva et al. 2011) and zinc finger nucleases (ZFN) (Urnov et al. 2005, 2010), and later Transcription Activator-Like Effector Nucleases (TALEN) (Boch et al. 2009; Moscou and Bogdanove 2009; & Haoyi Wang ; CRISPRCas9-mediated genome editing The CRISPRCas system was first described in the genome of Escherichia coli as a cluster of short palindromic repeats separated by peculiar short spacer sequences (Ishino et al. 1987). Subsequently, it was shown that CRISPR loci are present in the genomes of more than 40 % of bacteria and 90 % of archaea (Horvath and Barrangou 2010) and their function is to serve as an adaptive immune defense mechanism, protecting against phage infection by recognizing and cleaving pathogen DNA (Horvath and Barrangou 2010; Fineran and Charpentier 2012). By 2012, the basic mechanism of CRISPRCas9 derived from Streptococcus pyogenes was elucidated (Deltcheva et al. 2011; Jinek et al. 2012). CRISPRCas9 is an RNA-guided DNA endonuclease system in which Cas9 endonuclease forms a complex with two naturally occurring RNA species, CRISPR RNA (crRNA) and trans activating CRISPR RNA (tracrRNA). This complex targets specific DNA sequences complementary to the 20 nt (nucleotide) sequence residing at the 50 end of the crRNA (Jinek et al. 2012). Conveniently, crRNA and tracrRNA can be linked by an arbitrary stem loop sequence to generate a synthetic singleguide RNA (sgRNA). Although naturally evolving as a system in bacteria, upon appropriate codon optimization of the Cas9 coding sequence, CRISPRCas9 is highly active in mammalian cells (Cho et al. 2013; Cong et al. 2013; Jinek et al. 2013; Mali et al. 2013b). In practice, by simply designing the 50 20 nt sequence on the sgRNA to be complementary to the genomic target sequence, the Cas9 nuclease-sgRNA complex can be directed to specific genomic locus generating DNA DSBs. The target defining region of the sgRNA is about 20 nt long, with variations from 17 to 30 nt having been successfully used (Ran et al. 2013; Fu et al. 2014). The other key element in determining target sequence specificity is the Protospacer Adjacent Motif (PAM) that is adjacent to the target site at the genome locus, but is not a part of the guide RNA sequence (see Fig. 1). For Cas9 nuclease from S. pyogenes, the PAM sequence is NGG, while CRISPR Cas9 systems from other species use different PAM sequences (Cong et al. 2013; Esvelt et al. 2013; Hou et al. 2013). In bacteria, the PAM is thought to effectively distinguish self, with the PAM not being present in the genomic CRISPR loci, from the invading phage, whose genome carries the PAM sequence adjacent to the target sequence (Marraffini and Sontheimer 2010). CRISPRCas9-mediated DNA DSBs are repaired through either the Non-Homologous End Joining (NHEJ) repair process, or the homology-directed repair (HDR) pathway. NHEJ repair often leads to small insertions or deletions (indels) at the targeted site, while HDR pathway leads to perfect repair or precise genetic modification (see Fig. 1) (Doudna and Charpentier 2014; Hsu et al. 2014). Through these two DNA repair pathways, various genetic modifications can be achieved (Fig. 1). The NHEJ-mediated DNA repair pathway can be exploited to generate null mutation alleles. Indel mutations generated at a target site within an exon can lead to frame shift mutations in one or both alleles. One major advantage of the CRISPRCas9 system, as compared to conventional gene targeting and other programmable endonucleases, is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene (Wang et al. 2013a, b). In addition, when two sgRNAs are used flanking a genomic region, the intervening region can be deleted or inverted (Blasco et al. 2014; Canver et al. 2014; He et al. 2015). Furthermore, chromosomal translocation can also be achieved by using two sgRNAs targeting two genomic loci located on different chromosomes (Choi and Meyerson 2014). Fig. 1 CRISPRCas9-mediated genome editing. a The structure of Cas9sgRNA complex binding to target DNA. Cas9 binds to specific DNA sequences via the base-pairing of the guide sequence on sgRNA (pink) with the DNA target (gray). Protospacer adjacent motif (PAM) is downstream of the target sequence. b The CRISPRCas9mediated double-stranded DNA breaks are repaired by endogenous DNA repair machinery: non-homologous end joining (NHEJ) or homology-directed repair (HDR). Various genetic modifications can be generated through these two pathways When a DSB is generated and a donor DNA template is provided, precise genetic modification can be introduced through the HDR pathway (Fig. 1). For small modifications, including incorporation of point mutations, defined indel mutations, as well as insertion of a short sequence such as a loxP site or an epitope tag, single-stranded oligodeoxynucleotide (ssODN) can be used as donor DNA. In this design, donor ssODN is designed to carry homologous sequences flanking the mutation and total size can be up to 200 nt. HDR efficiency does not appear to be directly correlated with donor homology lengths (Yang et al. 2013b), and HDR efficiency variation is likely due to the nature of the target genomic loci, which is still poorly understood. When DNA of larger sizes is to be introduced into a target site, a double-stranded donor plasmid carrying the transgene flanked by homologous arms is used (Yang et al. 2013a). Because of the ease of use, CRISPRCas9 system has swiftly become the most commonly used tool for efficient genome editing of bacteria, plants, cell lines, primary cells, a (...truncated)