Genome engineering using the CRISPR-Cas9 system (original) (raw)

The ability to engineer biological systems and organisms holds enormous potential for applications across basic science, medicine and biotechnology. Programmable sequence-specific endonucleases that facilitate precise editing of endogenous genomic loci are now enabling systematic interrogation of genetic elements and causal genetic variations 1,2 in a broad range of species, including those that have not previously been genetically tractable 3-6. A number of genome editing technologies have emerged in recent years, including zinc-finger nucleases (ZFNs) 7-10 , transcription activator-like effector nucleases (TALENs) 10-17 and the RNA-guided CRISPR-Cas nuclease system 18-25. The first two technologies use a strategy of tethering endonuclease catalytic domains to modular DNA-binding proteins for inducing targeted DNA double-stranded breaks (DSBs) at specific genomic loci. By contrast, Cas9 is a nuclease guided by small RNAs through Watson-Crick base pairing with target DNA 26-28 (Fig. 1), representing a system that is markedly easier to design, highly specific, efficient and well-suited for highthroughput and multiplexed gene editing for a variety of cell types and organisms. Precise genome editing using engineered nucleases Similarly to ZFNs and TALENs, Cas9 promotes genome editing by stimulating a DSB at a target genomic locus 29,30. Upon cleavage by Cas9, the target locus typically undergoes one of two major pathways for DNA damage repair (Fig. 2): the error-prone NHEJ or the high-fidelity HDR pathway, both of which can be used to achieve a desired editing outcome. In the absence of a repair template, DSBs are re-ligated through the NHEJ process, which leaves scars in the form of insertion/deletion (indel) mutations. NHEJ can be harnessed to mediate gene knockouts, as indels occurring within a coding exon can lead to frameshift mutations and premature stop codons 31. Multiple DSBs can additionally be exploited to mediate larger deletions in the genome 22,32. HDR is an alternative major DNA repair pathway. Although HDR typically occurs at lower and substantially more variable frequencies than NHEJ, it can be leveraged to generate precise, defined modifications at a target locus in the presence of an exogenously introduced repair template. The repair template can either be in the form of conventional double-stranded DNA targeting constructs with homology arms flanking the insertion sequence, or single-stranded DNA oligonucleotides (ssODNs). The latter provides an effective and simple method for making small edits in the genome, such as the introduction of singlenucleotide mutations for probing causal genetic variations 32. Unlike NHEJ, HDR is generally active only in dividing cells, and its efficiency can vary widely depending on the cell type and state, as well as the genomic locus and repair template 33. Cas9: an RNA-guided nuclease for genome editing CRISPR-Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements 18-21,26. Three types (I-III) of CRISPR systems have been identified across a wide range of bacterial and archaeal hosts, wherein each system comprises a cluster of CRISPR-associated (Cas) genes, noncoding RNAs and a distinctive array of repetitive elements (direct repeats). These repeats are interspaced by short variable sequences 20 derived from exogenous DNA targets known as protospacers, and together they constitute the CRISPR RNA (crRNA) array. Within the DNA target, each protospacer is always associated with a protospacer adjacent motif (PAM), which can vary depending on the specific CRISPR system 34-36. The Type II CRISPR system is one of the best characterized 26-28,37,38 , consisting of the nuclease Cas9, the crRNA array that encodes the guide RNAs and a required auxiliary trans-activating crRNA (tracrRNA) that facilitates the processing of the crRNA array into discrete units 26,28. Each crRNA unit then contains a 20-nt guide sequence and a partial direct repeat, where the former directs Cas9 to a 20-bp DNA target via Watson-Crick base pairing (Fig. 1). In the CRISPR-Cas system derived from Streptococcus pyogenes (which is the system used in this protocol), the target DNA must immediately precede a 5′-NGG PAM 27 , whereas