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.
Highlights
While 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
Framework for fair evaluation of different CRISPR-Cas systems We sought to establish an evaluation framework that allowed an unbiased assessment of the five Cas endonucleases (SpCas9, smaller Cas9 enzymes from Staphylococcus aureus (SaCas9), NmCas9, AsCpf1, and LbCpf1)
Since each Cas nuclease may be differentially affected by chromatin accessibility, we targeted genes with varying expression levels in HEK293T cells because gene transcription is largely controlled by the underlying chromatin architecture [21]
Summary
While 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. The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system is a versatile tool that has been successfully used to modify the genome of myriad organisms [1,2,3,4,5,6,7,8,9,10] In this system, an endonuclease (typically Cas or Cpf1) is recruited to a specific genomic locus by a chimeric single guide RNA (sgRNA), which comprises both a crRNA spacer that recognizes the target site by reverse complementary base pairing and a stem loop-containing scaffold for the nuclease [11,12,13]. By incorporating various CRISPR-Cas systems into our engineering toolbox, we can expand the range of sites to target in a genome
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.