It is widely accepted that the lion's share of gene editing advances enabled by CRISPR based gene editing have come from the discovery and utilisation of class 2 CRISPR-Cas effectors. Diverse lineages of class 2 effector proteins possess several generalisable properties, which grant them a comparative advantage in gene editing and knockdown applications compared with class 1 systems. Class 2 effector proteins are monomeric and sequence specifically degrade DNA at a single loci within the target genome or transcriptome upon binding, usually introducing either a single or double strand break in the target nucleic acid strand. This avoids the possible side effects arising from degrading a large DNA region as is the case in type Ⅰ CRISPR-Cas effector mediated gene editing[56–57]. As a consequence of these properties, the design and operation of computational pipelines, to extract and characterise the full extent of class 2 effectors diversity has been a major priority in the last 5 years. This has unveiled 3 basic types of class 2 effectors (Fig. 3B), subclassified into a plenitude of subtypes with the potential to complement or surpass the traditional first generation SpCas9-CRISPR mediated gene editing platform.
It is indisputable that the breakthrough generated from the utilisation of Streptococcus pyogenes Cas9 (SpCas9) to induce programmable RNA-guided Double Stranded cleavage has since become the epicentre of the CRISPR-Cas gene-editing world. Cas9 distinguishes itself from other effector types by its high abundance (present in approximately 10% of bacteria), distinct mechanism of double stranded cleavage (Fig. 6A), high efficacy in diverse organisms and relatively low restrictions on programmability due to small PAM requirements[76–77]. While other type Ⅴ effectors utilise a single RuvC domain to cleave both DNA strands, Cas9 uses its RuvC and HNH domains to cleave the complementary and non target strands almost simultaneously. This cleavage is specific to the target site, with no indiscriminate single stranded DNA (ssDNA) cleavage occurring as a side reaction, which is often observed with the effectors of Type Ⅴ systems such as Cas12a.
Due to being the first ortholog discovered, there has been a much greater exploration of Cas9 ortholog diversity and re-engineering of successful orthologs into higher activity variants than for effectors from other types (Table 1)[27,46–47,81,101–103]. The driving motivation for using different Cas9 orthologs for editing lies in their different PAM requirements, protein size to fit into a delivery vector and editing efficacy at different target sites in different organisms. Both Staphylococcus aureus Cas9 (SaCas9, 1053 residues) and Campylobacter jejuni Cas9 (CjeCas9, 984 residues) are smaller than SpCas9, the standard effector used for most editing applications[46,81]. This results in a smaller construct size when genes encoding either of these effector proteins are cloned onto an insertion vector for gene delivery Certain orthologs may also provide an efficacy and specificity improvement when used at certain target sites, due fewer possible off-target sites due to the effector's PAM being more specific to the target site of interest[82–83]. Although overall, there has been relatively little success in finding a naturally occurring Cas9 ortholog, which surpasses SpCas9 in terms of functionality for general purpose use, when used as a collective toolbox for a specific target site, the utilisation of these alternative Cas9 orthologs can significantly increase the specificity and efficacy of the editing reaction, as well as the number of possible sites to induce cleavage in a gene of interest.
Abbreviation Species Size (aa) PAM gRNA size (bp) crRNA (5′→3′) tracrRNA (5′→3′) Reference spCas9 Streptococcus Pyogenes 1368 NGG 20 GUUUUAGAGCUAUGCUGUUUUG GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU Jinek et al, (2012) FnCas9 Francisella novicida 1629 NGG 20 CUAACAGUAGUUUACCAAAUAAUUCAGCAACUGAAAC GUUGUUAGAUUAUUUGGUAUGUACUUGUGUUAGUUUAAAGUAGCUAGAAAAUUCACUUUUAGACCUACUUAUUUUU Sampson et al, (2013)[47,101] SaCas9 Staphylococcus aureus 1053 NNGRRT 21 GUUUUAGUA CUCUGUAAU UUUAGGUAU GAGGUAGAC AUUGUACUUAUACCUAAAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUU Ran et al, (2015) NmCas9 Neisseria meningitidis
1082 NNNNGATT 24 NGUUGUAGCUCCCUUUCUCAUUUCG AAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCGCAACGCUCUGCCCCUUAAAGCUUCUGCUUUAAGGGGCAUCGUUUA Hou et al, (2013)[101-102] St1Cas9 Streptococcus thermophilus
1121 NNAGAAW 20 GUUAUUGUACUCUCAAGAUUUAUUUUU GUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUGUUUUCGUUAUUUAA Gasiunas et al, (2012); Cong et al, (2013)[27,101,103] St3Cas9 Streptococcus thermophilus
1409 NGGNG 20 GUUCGUACUUAGUUUUAGAGCUGUGUUGUUUCG GUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCYGUCAUUUUAUGGCAGGGUGUUUUCGUUAUUUAA Gasiunas et al, (2012); Cong et al, (2013)[27,101,103] CjCas9 Campylobacter jejuni 984 NNNNACAC 22 GUUUUAGUCCCUUUUUAAAUUUCUUUAUGGUAAAU AAGAAAUUUAAAAAGGGACUAAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU Kim et al, (2017)[81,101]
Table 1. Properties of most commonly used Cas9 orthologs in gene editing
Although all Cas12 effectors possess and utilise a single RuvC domain for nucleic acid strand cleavage, the substrate requirements and mechanism of cleavage differ substantially between different effector types(Fig. 6B-F). To date there are 11 different known Cas12 effector types, alphabetised A to K (Fig. 3B). The mechanism underlying this cleavage has predominantly been studied in Cas12a orthologs, but provides some transferable insight into the process for other Cas12 effectors as well[85–88]. For Cas12a, RNA-DNA heteroduplex formation induces conformational change in the NUC lobe, making the RuvC catalytic residues accessible. Cleavage of the non-target strand must precede cleavage of the target strand. This results in a staggered cleavage pattern with approximately 5 nt 5′ overhangs (Fig. 6B). This releases the PAM-distal target DNA fragment. However, the ribonucleoprotein complex is still catalytically competent while bound to the PAM-proximal DNA[89–90]. This often results in the activation of a secondary activity wherein indiscriminate cleavage, or 'trans' cleavage of ssDNA (and in some orthologs single stranded RNA (ssRNA), and nicking of dsDNA) by the effector protein occurs[79,89].
One recent advance has been the discovery and characterisation of Cas12j (phi) effectors. These proteins, encoded exclusively on the genomes of large phages and more compact (700 to 800 aa in size) than other Cas12 effectors. While these effectors possess dsDNA cleavage activity, there was a significant difference in efficacy between Cas12j mediated cleavage of the target strand, and Cas12a mediated cleavage of the target strand[38,39,91–92]. This shortcoming means that while the characterisation of Cas12j represents an important step towards more compact, high efficacy editing proteins, it is however unlikely to supersede existing editors such as Cas12a or Cas9.
One of the most exciting potential advances arising from the exploration of CRISPR-Cas effector diversity has been the discovery of tiny, 400 to 700 amino acids long effector proteins. These proteins are small enough to be delivered in a recombinant adenoviral vector (rAAV). Unfortunately, all Cas14 effectors discovered to date cleave ssDNA with relatively high efficacy, but are unable to cleave dsDNA with comparable efficacy which limits their potential application without protein engineering optimization (Fig. 6E)[39,91]. Nevertheless, there is considerable optimism that this limitation can be surmounted either via direct protein engineering of Cas14 to produce a gain of function variant with higher cleavage activity, or via further exploration and characterization of Cas14 orthologs.
A subclade of Cas12 effectors exist that lack functional RuvC catalytic residues and occur in the same operon as Tn7-like transposases. These function as RNA-guided DNA binding proteins and form a complex with Tn7 transposases to direct the site of transposon integration (Fig. 6D). Compared with type Ⅰ-Tn7 transposon integration systems, Cas12k guided systems offer two important distinct advantages in the form of higher insertion efficacy, depending on the loci chosen for targeting, and simpler and smaller construct size, due to the monomeric nature of Cas12k effectors compared with type Ⅰ-F and Ⅰ-B Cascade surveillance complexes. The efficacy of integration by Cas12k proteins (in the range of 15% to 65% in E. Coli) is superior to the measured efficacy in yeast and eukaryotes of prime editing, an alternate means of integrating DNA in host genomes. This efficacy is also competitive with the editing efficacy of Cas9 or Cas12a without the side-effects associated with the utilisation of DSB repair pathways for editing, although further research is needed to demonstrate feasibility in eukaryotic cell lines[94–96].
A different avenue for bypassing the limitations of DSB based gene knockout protocols is to induce a gene knockdown at the target site. This involves utilizing RNA-guided site-specific riboendonucleases to target and cleave mRNA transcribed by the gene of interest[14,37,38,97–98]. This silences the expression of the target gene. Several Cas12 orthologs have been discovered which possess RNA-guided RNAse activity (Fig. 6C and E), and an entire clade of CRISPR-Cas effectors, designated Cas13, have been discovered which exclusively target and cleave single strand RNA in a manner analogous to RNA-guided DNA targeting CRISPR-Cas effectors[14,37–98–99].
Harnessing CRISPR-Cas system diversity for gene editing technologies
- Received Date: 2020-11-12
- Accepted Date: 2021-02-19
- Rev Recd Date: 2021-02-05
- Available Online: 2021-03-26
- Publish Date: 2021-03-26
- CRISPR-Cas systems /
- gene editing /
- biological evolution /
- DNA repair /
- classification /
- DNA transposable elements
Abstract: The discovery and utilization of RNA-guided surveillance complexes, such as CRISPR-Cas9, for sequence-specific DNA or RNA cleavage, has revolutionised the process of gene modification or knockdown. To optimise the use of this technology, an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements, coupled with high cleavage efficacy and specificity. Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.
|Citation:||Alexander McKay, Gaetan Burgio. Harnessing CRISPR-Cas system diversity for gene editing technologies[J]. The Journal of Biomedical Research, 2021, 35(2): 91-106. doi: 10.7555/JBR.35.20200184|