Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

Abstract

BackgroundWhile CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing.ResultsWe systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform precise gene targeting efficiently across multiple genomic loci using single-stranded oligodeoxynucleotide (ssODN) donor templates with homology arms as short as 17 nucleotides. Strikingly, the two Cpf1 nucleases exhibit a preference for ssODNs of the non-target strand sequence, while the popular Cas9 enzyme from Streptococcus pyogenes (SpCas9) exhibits a preference for ssODNs of the target strand sequence instead. Additionally, we find that the HDR efficiencies of Cpf1 and SpCas9 can be further improved by using asymmetric donors with longer arms 5′ of the desired DNA changes.ConclusionsOur work delineates design parameters for each CRISPR-Cas system and will serve as a useful reference for future genome engineering studies.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1445-x) contains supplementary material, which is available to authorized users.

A simple and efficient workflow for generation of knock‐in mutations in Jurkat T cells using CRISPR/Cas9

“…Accounting also for ‘CC’ (representing the PAM site on the complementary strand) will give on average one PAM site within a 10 base distance from the mutation site of interest. Although PAM site recognized by Cas9 from S pyogenes is relatively simple and abundant, it might be advantageous to use CRISPR systems from other bacteria, which recognize other PAM sites . It could potentially allow cutting of DNA closer to the target site, and increase the efficiency of HDR.…”

“…Although PAM site recognized by Cas9 from S pyogenes is relatively simple and abundant, it might be advantageous to use CRISPR systems from other bacteria, which recognize other PAM sites. 35 It could potentially allow cutting of DNA closer to the target site, and increase the efficiency of HDR.…”

Clinical applications of CRISPR‐based genome editing and diagnostics

“…At this stage, genetic knockouts are facile, but replacing sequences by HDR remains inefficient and technically challenging, although great strides are being made to improve the technique. [65][66][67][68][69][70][71][72] CRISPR systems have generated an extraordinary level of excitement for the clinical potential for curing human disease. In this nascent stage of CRISPR in the clinic, initial applications are treating diseases with the most accessible cells (immune cells and HSPCs edited ex vivo), which avoids the ongoing challenge of tissue-specific delivery in vivo.…”

“…54 The donor is typically a singleor double-stranded DNA bearing "homology arms" that match either side of the Cas9 cut site. 65 The likelihood of successful insertion can be improved using a few different strategies, but the general inefficiency of this gene replacement approach represents a substantial challenge in the field. [65][66][67][68][69][70][71][72] As our ability to carefully manipulate DNA grows, so will the clinical potential of CRISPR technologies.…”

“…65 The likelihood of successful insertion can be improved using a few different strategies, but the general inefficiency of this gene replacement approach represents a substantial challenge in the field. [65][66][67][68][69][70][71][72] As our ability to carefully manipulate DNA grows, so will the clinical potential of CRISPR technologies. 17 CURRENT AND NEAR-TERM THERAPEUTIC APPLICATIONS Genetic therapies offer hope for treating and effectively curing monogenic diseases by reversing the underlying genetic cause.…”