Original researchHarnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon
Introduction
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are present in ∼40% of bacterial and ∼90% of archaeal genomes, and provide adaptive immunity against foreign genetic elements (Barrangou et al., 2007, Sorek et al., 2008, Barrangou and Marraffini, 2014). CRISPR-Cas systems are highly diversified and currently classified into 2 classes (class 1 and 2), 6 types (type I–VI) and at least 27 subtypes (Makarova et al., 2015, Shmakov et al., 2015, Shmakov et al., 2017). CRISPR immunity involves three functional stages: adaptation, expression, and interference stage (van der Oost et al., 2009, Marraffini and Sontheimer, 2010, Westra et al., 2014). During the adaptation stage, foreign DNA fragments (namely protospacers) from invading genetic elements are acquired and incorporated into the CRISPR array as new spacer sequences (Sternberg et al., 2016). During the expression stage, the CRISPR array is transcribed into precursor RNA, which is processed into mature CRISPR RNAs (crRNAs) (Brouns et al., 2008; Carte et al., 2010; Haurwitz et al., 2010; Li et al., 2013). During the final interference stage, the crRNA spacer specifically matches the protospacer sequence on the invader DNA/RNA and elicits target destruction with the help of Cas nuclease(s) (Marraffini and Sontheimer, 2008, Hale et al., 2009; Garneau et al., 2010). For type I and II systems, “target” versus “non-target” discrimination during interference is achieved by sensing the protospacer adjacent motif (PAM) (Westra et al., 2013, Li et al., 2014b). Next to the PAM, there is a 7–12 bp seed region where spacer-protospacer base paring is particularly important for interference (Wiedenheft et al., 2011, Semenova et al., 2011, Plagens et al., 2015).
DNA double-strand breaks (DSBs) caused by CRISPR-mediated cleavage can be repaired by homology-directed repair (HDR) or nonhomologous end-joining (NHEJ), which can be utilized to achieve genome editing. Type II system-mediated interference is simpler, because it only requires a multifunctional protein Cas9, which is directed by the dual-tracrRNA:crRNA or a synthetic RNA chimera to cleave the target DNA (Deltcheva et al., 2011, Jinek et al., 2012). The powerful CRISPR/Cas9 genome editing technique has been widely employed for genetic manipulation in diverse organisms (DiCarlo et al., 2013, Jiang et al., 2013, Shan et al., 2013, Wang et al., 2013) or human cells (Cong et al., 2013, Mali et al., 2013). Recently, the class 2 CRISPR effector protein Cpf1 has been reported to be a single-RNA-guided endonuclease and has been exploited for genome editing in several human cells (Zetsche et al., 2015), in which case the off-target effect was shown to be below the detectable level (Kim et al., 2016, Kleinstiver et al., 2016).
For genome editing in prokaryotes, an alternative strategy is to utilize their native CRISPR-Cas systems. For example, the type I and III systems in the thermophilic archaeon Sulfolobus islandicus were separately exploited for genome editing (Li et al., 2016). In the bacterium Clostridium pasteurianum, a high editing efficiency was recently reported for its native type I-B CRISPR-Cas (Pyne et al., 2016). However, intriguingly, in the halophilic archaeon Haloferax volcanii, I-B CRISPR-mediated self-targeting was shown to be highly tolerated (Stachler et al., 2017), which impedes this CRISPR system from being exploited. It was suggested that the high chromosome copy number (over 20 copies per cell) in halophilic archaeal cells (Breuert et al., 2006, Zerulla et al., 2014), which facilitates rapid and accurate repair via homologous recombination (HR), may be responsible for this high tolerance.
Like all known haloarchaea, the haloarchaeon Haloarcula hispanica is also polyploid (Fig. S1). It has been an important model for studying archaeon-virus interactions, which has provided the first archaeal CRISPR-Cas system showing efficient adaptation to a purified virus (Li et al., 2014a). The three CRISPR functional stages, especially the adaptation stage, have been extensively characterized by our lab (Li et al., 2013; Li et al., 2014a, Li et al., 2014b, Li et al., 2017, Wang et al., 2016). Notably, this CRISPR system mediates active interference against a target DNA (Li et al., 2014a, Li et al., 2014b), which facilitates repurposing for genome editing. In this study, we report that self-targeting mediated by the H. hispanica CRISPR-Cas is highly cytotoxic to the host, which seems different from the situation in H. volcanii. Our results also demonstrated that in this polyploid archaeon, precise genome editing, including gene deletion, gene tagging and single nucleotide substitution could be readily achieved using the CRISPR-based genome-editing pipeline. Technically, the CRISPR strategy shows a much higher editing efficiency (especially when multiple genome loci need to be edited) compared to the traditional pyrF-based double-selection strategy.
Section snippets
Exploitation of type I-B CRISPR for gene-knockout in H. hispanica
The native H. hispanica CRISPR-Cas system (Fig. 1A) is able to mediate active interference against a re-infecting virus or a target plasmid, which results in a PFU (plaque forming unit) reduction of 6–7 orders of magnitude (Li et al., 2014a) or a CFU (colony forming unit) reduction of ∼3 orders of magnitude (Li et al., 2014b). In addition to the spacer-protospacer homology, the PAM sequence 5′-TTC-3′ immediately upstream of the protospacer is also critical for CRISPR-mediated DNA cleavage (Li
Discussion
Exploiting the native CRISPR-Cas system for genome editing has been demonstrated to be convenient in the bacterium C. pasteurianum (Pyne et al., 2016) and the thermophilic archaeon S. islandicus (Li et al., 2016), both of which are monoploid (containing one chromosome copy per cell). Interestingly, a recent attempt to harness the native CRISPR-Cas for precise genome editing in H. volcanii, one of the halophilic archaea that have been reported to be polyploid (containing multiple chromosome
Strains, culture conditions, plasmids, and primers
The H. hispanica strains used in this study are listed in Table S1. The DF60 strain (a uracil auxotrophic ΔpyrF mutant of H. hispanica ATCC 33960) and its derivatives were cultured at 37°C in nutrient-rich AS-168 medium (200 g of NaCl, 2 g of KCl, 20 g of MgSO4·7H2O, 3 g of trisodium citrate, 1 g of sodium glutamate, 5 g of Bacto casamino acids, 5 g of yeast extract, 50 mg of FeSO4·7H2O, and 0.36 mg of MnCl2·4H2O per liter, pH 7.2) with uracil at a final concentration of 50 μg/mL. When needed,
Acknowledgments
This work was supported by the grants from the National Natural Science Foundation of China (No. 31571283) and by the CAS-SAFEA International Partnership Program for Creative Research Teams.
References (49)
- et al.
CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity
Mol. Cell
(2014) - et al.
An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA
J. Biol. Chem.
(2012) - et al.
RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex
Cell
(2009) - et al.
Characterization of CRISPR RNA biogenesis and Cas6 cleavage-mediated inhibition of a provirus in the haloarchaeon Haloferax mediterranei
J. Bacteriol.
(2013) - et al.
Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica
J. Genet. Genomics
(2011) - et al.
An active immune defense with a minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and without the Cas6 protein
J. Biol. Chem.
(2015) - et al.
Discovery and functional characterization of diverse class 2 CRISPR-Cas systems
Mol. Cell
(2015) - et al.
Adaptation in CRISPR-Cas systems
Mol. Cell
(2016) - et al.
CRISPR-based adaptive and heritable immunity in prokaryotes
Trends biochem. Sci.
(2009) - et al.
One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering
Cell
(2013)