Hence, the PAM places a constraint on the genomic locations where a particular Cas nuclease is able to cleave. The Cas-guide RNA complex is unable to introduce a DSB if the PAM is absent 17. The PAM of Cas9 is at the 3вЂ™ end of the protospacer, while the PAM of Cas12a (also called Cpf1) is at the 5вЂ™ end instead 16. The endonuclease activity of the Cas protein requires the presence of a protospacer adjacent motif (PAM) at the target site 15. The former is typically used if the intended DNA changes are small (for example, alteration of a single base pair), while the latter is typically used if one wishes to insert a relatively long sequence (for example, the coding sequence of a green fluorescent protein or GFP) into the target locus. Common types of donor templates include single-stranded oligonucleotides (ssODNs) and plasmids. However, in the presence of a donor template that contains the desired DNA changes, the DSB is repaired by the high fidelity HDR pathway. In the absence of a repair template, the DSB is repaired by the error-prone NHEJ pathway, which can give rise to pseudo-random insertion or deletion of nucleotides (indels) at the cut site, potentially causing frameshift mutations in protein-coding genes. The resulting double stranded break (DSB) is subsequently repaired by the cells via either the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway. After being recruited to the target site, the Cas enzyme functions as a pair of molecular scissors and cleaves the bound DNA with its RuvC, HNH, or Nuc domains 12, 13, 14. A primary reason why CRISPR-Cas has gained so much popularity in such a short time is that the element that brings the key Cas endonuclease, such as Cas9 or Cas12a (also known as Cpf1), to the correct location in the genome is simply a short piece of chimeric single guide RNA (sgRNA), which is straightforward to design and cheap to synthesize. Originally an adaptive immune system in bacteria, it has been successfully deployed for genome modification in plants and animals, including humans. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated (Cas) system has emerged as a powerful new genome engineering technology 10, 11.

However, these earlier technologies are either difficult to program or tedious to assemble, thereby hampering their widespread adoption in research and the industry.

Since the turn of the century, various technologies have been developed for genome engineering in mammalian cells, including meganucleases 1, 2, 3, zinc finger nucleases 4, 5, or transcription activator-like effector nucleases (TALENs) 6, 7, 8, 9. The ability to engineer the genome of any living organism has many biomedical and biotechnological applications, such as the correction of disease-causing mutations, construction of accurate cellular models for disease studies, or generation of agricultural crops with desirable traits. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms.

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