Fbioe 08 00711

REVIEW
published: 30 June 2020

doi: 10.3389/fbioe.2020.00711
Development and Application of

CRISPR/Cas in Microbial
Biotechnology
Wentao Ding 1,2 , Yang Zhang 1 and Shuobo Shi 1*
1
Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology,
Beijing, China, 2 Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering
and Biotechnology, Tianjin University of Science and Technology, Tianjin, China
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated

(Cas) system has been rapidly developed as versatile genomic engineering tools with
high efficiency, accuracy and flexibility, and has revolutionized traditional methods for
applications in microbial biotechnology. Here, key points of building reliable CRISPR/Cas
system for genome engineering are discussed, including the Cas protein, the guide RNA
and the donor DNA. Following an overview of various CRISPR/Cas tools for genome
engineering, including gene activation, gene interference, orthogonal CRISPR systems
and precise single base editing, we highlighted the application of CRISPR/Cas toolbox
Edited by: for multiplexed engineering and high throughput screening. We then summarize recent
Yi Wang,
applications of CRISPR/Cas systems in metabolic engineering toward production of
Auburn University, United States
chemicals and natural compounds, and end with perspectives of future advancements.
Reviewed by:
Yuan Qiao, Keywords: CRISPR/Cas, guide RNA, genome editing, gene regulation, microbial biotechnology
Nanyang Technological University,
Singapore
Mingfeng Cao,
University of Illinois
INTRODUCTION
at Urbana-Champaign, United States
Microbial cell factories producing fuels, chemicals, and pharmaceutics are perspective production
*Correspondence:
mode to replace petrol relied methods because microbial methods are usually clean and renewable.
Shuobo Shi
shishuobo@mail.buct.edu.cn
One restriction to the development of microbial producer is the slow, inefficient and arduous
genomic engineering processes. The emerging toolbox based on clustered regularly interspaced
Specialty section: short palindromic repeats (CRISPR) system have largely improved genome editing efficiency,
This article was submitted to simplified steps of multi-loci editing, and enabled fast disturbance of metabolic network. The
Synthetic Biology, CRISPR system is prokaryotic adaptive immune system against intruded heterologous DNA/RNA
a section of the journal from virus or other organisms (Grissa et al., 2007a; Sorek et al., 2013). So far, the CRISPR/Cas
Frontiers in Bioengineering and system has been intensively adopted as toolbox for both fundamental studies and biotechnological
Biotechnology
applications for genome editing, molecular diagnosis, metabolic engineering, gene function mining,
Received: 16 April 2020 etc., in microorganisms, plants and mammals (Sander and Joung, 2014; Zhang et al., 2014; Wang
Accepted: 08 June 2020
H. et al., 2016; Tang and Fu, 2018; Tarasava et al., 2018; Armario Najera et al., 2019; Moon et al.,
Published: 30 June 2020
2019; Xu and Oi, 2019). In the field of microbial biotechnology, the CRISPR/Cas system has been
Citation: applied for numerous model and non-model microorganisms, e.g., Escherichia coli (Jiang et al.,
Ding W, Zhang Y and Shi S (2020)
2013), Saccharomyces cerevisiae (DiCarlo et al., 2013), Bacillus (Westbrook et al., 2016), Clostridium
Development and Application
of CRISPR/Cas in Microbial
(Li et al., 2016; Joseph et al., 2018), Corynebacterium (Jiang et al., 2017), Lactobacillus (Oh and van
Biotechnology. Pijkeren, 2014), Mycobacterium (Choudhary et al., 2015), Pseudomonas (Tan S. Z. et al., 2018),
Front. Bioeng. Biotechnol. 8:711. Streptomyces (Cobb et al., 2015). However, there still remains interested microorganisms that
doi: 10.3389/fbioe.2020.00711 CRISPR system has not been applied, and some weakness of existing CRISPR/Cas systems needs
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 June 2020 | Volume 8 | Article 711
Ding et al. Application of CRISPR in Microorganism
to be overcome. This review focuses on the establishment and at DNA target near PAM (Mougiakos et al., 2016). The spacer
development of CRISPR toolbox for genome editing and gene part is responsible for DNA target (also called protospacer)
regulation, and applications of these techniques in metabolic binding, and guides the Cas9 complex for sequence specific
engineering and synthetic biology in microorganisms. DNA cleavage. PAM flanks the 30 end of the protospacer, and is
required for Cas9-mediated cleavage (Deveau et al., 2008; Mojica
et al., 2009). The PAM of the most commonly used SpCas9 (Cas9
THE CRISPR/CAS SYSTEM FOR from Streptococcus pyogenes) is ‘NGG,’ which occurs once every
GENOME EDITING 8 bp on average within the genome, allowing targeting on most
genes of interest (Doudna and Charpentier, 2014; Hsu et al.,
The CRISPR systems are adaptive evolved for counteracting 2014). Cas9s from different resources recognize different PAM
foreign DNA or RNAs, and the systems are present in nearly half sequences, which further expands the application of CRISPR
of bacteria and almost all archaea (Grissa et al., 2007b; Zetsche for various genomic sequence [e.g., Cas9 from Staphylococcus
et al., 2015a), but absent from eukaryotes or viruses (Jansen et al., aureus (Kleinstiver et al., 2015; Ran et al., 2015), Streptococcus
2002). The CRISPR/Cas systems have been categorized into two thermophiles (Esvelt et al., 2013; Kleinstiver et al., 2015), Neisseria
classes and six major types based on the constitution of effector meningitides (Esvelt et al., 2013; Hou et al., 2013)]. Cas9 ‘nickase’
protein and signature genes, protein sequence conservation, and variant (nCas9), with mutations deactivating one nickase activity
organization of the respective genomic loci (Koonin et al., 2017; and converting the endonuclease activity of wildtype Cas9 to
Tang and Fu, 2018). Among these CRISPR systems, the Cas9 nickase activity, introduces a single stranded break (SSB) rather
(Type II), Cas12a (previously known as Cpf1, type V) and than DSB (Jinek et al., 2012; Cong et al., 2013). Generally,
their mutant variants are most investigated effectors, and have SSBs are repaired by HDR, not by NHEJ, thus nCas9 can
shown broad applicational potentials in genome editing, gene be applied for precise genome editing (Standage-Beier et al.,
regulation, DNA detection, DNA imaging, etc. (Tang and Fu, 2015). Another Cas9 mutant, the nuclease-deactivated Cas9
2018; Miao et al., 2019). (dCas9), has been fused with a variety of effectors, including
The CRISPR/Cas system can introduce a double-strand transcriptional activators, repressors, and epigenetic modifiers to
DNA break (DSB) at the specific DNA target (also called enable sequence specific genomic regulation (Gilbert et al., 2013,
protospacer) binding by a guide RNA (gRNA) and harboring 2014; Qi et al., 2013).
a short protospacer adjacent motif (PAM) flanked at the In 2013, the application of CRISPR/Cas9 system for genome
30 end of protospacer (Figures 1A,B; Garneau et al., 2010; editing was originally reported in human cells (Cong et al., 2013;
Gasiunas et al., 2012; Jinek et al., 2012; Wang H. et al., Jinek et al., 2013; Mali et al., 2013b), mouse cells (Cong et al.,
2016). A DSB triggers DNA repair through intrinsic cellular 2013), Zebrafish (Hwang et al., 2013), Saccharomyces cerevisiae
mechanisms, mainly including non-homologous end joining (DiCarlo et al., 2013), Streptococcus pneumoniae, and Escherichia
(NHEJ), which direct ligates two breaking ends with small coli (Jiang et al., 2013). In following studies, the CRISPR/Cas9
insertions or deletions (indels); and homology-directed system has been widely applied for genome editing in numerous
repair (HDR), which repair DSB according to a homologous microorganisms, plants and animals.
template (Hsu et al., 2014; Doetschman and Georgieva, As an eukaryotic model microorganism, S. cerevisiae was
2017). Considering the guide RNAs are easy to design and one of the earliest hosts for CRISPR/Cas9 mediated genome
expressed, Cas protein can be programmed to introduce editing (DiCarlo et al., 2013). In order to improve genome
DSBs at one or more DNA targets, making CRISPR/Cas editing efficiency, the Cas9 protein is usually highly expressed
an convenient and precise platform for genome editing by a strong constitutive promoter [e.g., TEF1 promoter (DiCarlo
(Doetschman and Georgieva, 2017). Compared with similar et al., 2013; Gilbert et al., 2013; Bao et al., 2015), TDH3
genome editing tools such as zinc-finger nucleases (ZFNs) promoter (Gilbert et al., 2013; Laughery et al., 2015; Jensen
(Kim et al., 1996; Urnov et al., 2010) and TAL effector et al., 2017)] in a episomal CEN low copy plasmid (DiCarlo
nucleases (TALENs) (Boch et al., 2009; Christian et al., 2010), et al., 2013; Gilbert et al., 2013) or episomal 2 µ high copy
CRISPR/Cas shows a significant advantage that it is easier to plasmid (Ryan and Cate, 2014; Bao et al., 2015; Shi et al., 2016;
target a specific region by adjusting a 20 nt spacer sequence Jensen et al., 2017). However, in some researches, expression
of gRNA, rather than producing target-specific proteins of Cas9 with strong promoter (e.g., promoter of TEF1, HXT7,
(Doetschman and Georgieva, 2017). and TDH3) showed toxic effect to cell growth (Ryan and Cate,
2014; Generoso et al., 2016). Nevertheless, medium strength
Selection and Expression of Cas Protein or weak promoters showed similar editing efficiency, and no
The CRISPR/Cas systems have been reported to have two classes significant negative impact on the strain’s growth rate. For
and six major types, and among these types, the class 2 type II efficient CRISPR editing rate, codon usage in heterologous
CRISPR system (CRISPR/Cas9) is currently most studied and organisms should be also considered to guarantee sufficient
developed as toolbox for gene editing and other applications. Cas9 abundance in vivo. In eukaryotes, Cas9 protein should
As shown in Figure 1A, the effector (Cas9) is activated when be transported to nuclei to facilitate genome editing, and thus
forming a complex with single guide RNA [sgRNA, a fusion the nuclear localization sequence (NLS) should be fused to
RNA of CRISPR targeting RNA (crRNA) and trans-activating the Cas9 protein (Figure 1B). In S. cerevisiae, the SV40 NLS
CRISPR RNA (tracrRNA) (Jinek et al., 2012)], and triggers DSB (‘PKKKRKV’) is typically fused to the N- or C-terminus of
FIGURE 1 | Guidelines for expression of Cas protein and sgRNA in CRISPR/Cas system. (A) Scheme of CRISPR/Cas9 system. The Cas9-sgRNA (or
Cas9-crRNA-tracrRNA) complex binds to DNA target arising from Watson-Crick base pairing of spacer sequence, and triggers double strand break (DSB) when next
to a short protospacer adjacent motif (PAM, ‘NGG’ for Cas9 from S. pyogenes). (B) Expression cassette for Cas9. For efficient targeting to nucleus in eukaryotes,
the Cas9 should be fused to NLS (nuclear localization sequence) at one end or both ends. (C) Scheme of CRISPR/Cas12a (Cpf1) system. Cas12a triggers DSB
through a similar scheme of Cas9, but depends on different PAM (‘NTTT’) and less folded crRNA, and creates a sticky end at 18–23 bases away from the PAM. (D)
Expression cassette for sgRNA. A promoter of RNA polymerase III (RNAP III) is usually required for directing sgRNA in nucleus and with less modification. A 20 bp
spacer should be well designed according to target DNA sequence for efficient editing rates and avoiding off-target effects. (E) Multi-sgRNA expression through
multi-cassettes. Repeated elements, such as promoters, gRNA scaffold and terminators are repeated for different spacer sequences. (F) Multi-sgRNA expression
through crRNA array and tracrRNA (HI-CRISPR system). Different spacers are separated with direct repeats (DRs) and expressed by one promoter of RNAP III. The
pre-crRNA is transcribed and processed into mature crRNA by RNase III and unknown nuclease(s). The tracrRNA and Cas9 protein are complexed with mature
crRNA to form the dual-RNA-guided nuclease. (G) gRNA multiplexing strategies. Both RNAP II and RNAP III promoter can be used for expression the sgRNA array,
where sgRNAs are separated by features for RNA cleavage. RNA endonuclease Csy4 recognizes a 28 nucleotide sequence flanking the sgRNA sequence and
cleaves after the 20th nucleotide. The hammerhead ribozyme and HDV ribozyme flanked the 50 and 30 of the sgRNA, respectively, allowing for self-cleaving
production of sgRNAs, which are not dependent on the presence of an exogenous protein. Polycistronic tRNA-gRNA architecture allows the production of multiple
sgRNAs by endogenous RNase P and RNase Z.
the Cas9, and two NLSs fused to one terminus or both were CRISPR/Cas9 system (Jiang et al., 2015). Thus, co-expression
also applicable. of heterologous phage-derived recombinase to improve the
The model bacteria E. coli has also been intensively researched frequency of homologous recombination showed significant
as a host for CRISPR/Cas9 mediated genome editing. However, improved survival rates when CRISPR/Cas9 and gRNA expressed
E. coli lacks the NHEJ mechanism for DSB repair (Chayot (Jiang et al., 2015; Pyne et al., 2015; Bassalo et al., 2016). In
et al., 2010), and is highly reliant on a native homology-directed E. coli, inducible promoters were mostly used for both Cas9 and
repair system with low efficiency, challenging the DSB producing gRNA expression.
The CRISPR/Cas9 system has also been constructed gRNA design can help to choose best gRNAs in various species
with similar strategy for non-model microorganisms (shown in Table 1). The rules for gRNA scoring includes
(Yan and Fong, 2017; Cho et al., 2018; Raschmanova et al., possible binding sites with mismatches in the spacer sequence
2018; Wang and Coleman, 2019). Generally, species-specific or in the seed sequence, the GC content and poly T presence
strong promoters should be used for Cas9 expression, either and self-complementarity (Heigwer et al., 2014; Liu et al.,
constitutively or inducible expressed. Codon optimization should 2015; Naito et al., 2015; Labun et al., 2019). Except for gene
also be conducted when the Cas9 protein cannot be efficiently editing, CRISPR-ERA and CHOPCHOP also help to design
expressed. In eukaryotic microorganisms, NLS should be fused to gRNAs for gene activation and repression (Liu et al., 2015;
Cas9 at one or both termini for cell nucleus localization. The NLS Labun et al., 2019).
of SV40 from S. cerevisiae has been proven effective and applied Generally, a strong expression of gRNA is recommended for
in other yeast species. Native DNA repair types and efficiency an efficient target binding and CRISPR complex activation.
also largely determined genome editing rate, because DSB To express RNA without modifications added by the
induced by CRISPR/Cas9 can be repaired by NHEJ, resulting in RNA polymerase II (RNAPII) transcription system, RNA
indels and gene inactivation, or be repaired by HDR, resulting in polymerase III (RNAPIII) regulatory elements have been
precise genome editing by supplying proper DNA donors. Thus, used for transcription of functional gRNA (Figure 1D), e.g.,
in some organisms with both NHEJ and HDR pathways, deletion the SNR52 promoter has been used in yeast (Raschmanova
of KU70/KU80 often repressed NHEJ and increased CRISPR et al., 2018) and U6 promoter has been used in human cells
mediate genome editing rate through HDR (Gao S. et al., 2016; (Zhang et al., 2014; Wang H. et al., 2016). However, it is
Schwartz et al., 2016; Cao et al., 2018; Bae et al., 2020). However, noted that some promoters require special rules of gRNA
in some organisms lacking HDR, phage-derived recombinases sequence, e.g., the U6 promoter or the T7 promoter require
(RecET and λ-Red) should be co-expressed with Cas9, similar a ‘G’ or ‘GG,’ respectively, at the 50 end of the RNA to be
to the approaches adopted in E. coli (Jiang et al., 2015; transcribed (Sander and Joung, 2014; Wang H. et al., 2016).
Wang B. et al., 2018). Despite RNAPIII promoters are suitable for gRNA transcription,
In addition to widely applied Cas9, Cas12a (also known as in some organisms, however, these promoters are poorly
Cpf1) is a newly emerging Cas protein that is currently under characterized. On the other hand, RNAPII promoters can
evaluation for gene editing potential (Zetsche et al., 2015a). also be used to express gRNAs when proper strategies are
Cas12a is a crRNA-guided endonuclease, lacking tracrRNA adopted (Nowak et al., 2016). A RNAPII promoter of rrk1
compared with Cas9, and cleaves DNA at 18 nucleotides away and its leader RNA was used to express sgRNA by flanking a
from the PAM, resulting in a DSB with 4- to 5-nucleotide Hammerhead ribozyme on the 30 end of gRNA (Figure 1G)
overhangs (Figure 1C; Zetsche et al., 2015a). Besides, Shmakov in fission yeast (Jacobs et al., 2014). Another research also
et al. (2015) further classified three class 2 CRISPR systems, used RNAPII promoter but flanked the sgRNA with a 28
including C2c1, C2c3, and C2c2, which further expands CRISPR nucleotide hairpin at each end that is recognized by the
toolbox for genome editing. endoribonuclease Csy4 (Figure 1G; Nissim et al., 2014).
Fusion gRNAs with a hammerhead (HH) ribozyme on their
Design and Expression of Guide RNA 50 end and a hepatitis delta virus (HDV) ribozyme on their
The efficient expression of guide RNA is also critical to a 30 end was also reported functional for RNAPII promoter
CRISPR system because the spacer sequence of guide RNA (Figure 1G; Nissim et al., 2014; Weninger et al., 2016).
is responsible for DNA target binding and thus decides the Interestingly, fusion of sgRNA with special RNA scaffold
editing loci, and is closely related to on-target and off-target (e.g., HDV, RNA triplex) would increase in vivo RNA stability
efficiency. Generally, one or more single guide RNAs (sgRNAs) and thus promote engineering efficiency (Nissim et al., 2014;
are expressed in a CRISPR/Cas system (Figures 1D–G); but in Ryan and Cate, 2014).
some other cases, a crRNA matrix and a tracrRNA, instead of When CRISPR/Cas system is constructed for multi-loci
sgRNAs, are expressed separately for efficient CRISPR editing editing (Figures 1E–G), several strategies have been proposed to
(Bao et al., 2015). The spacer sequence should be carefully enable an efficient expression of multiple gRNAs. Multi-sgRNA
designed, which binds to a DNA target close to a PAM sequence, expression could be achieved through multi-expression cassettes
and to promote editing efficiency and reduce off-target rate. using individual promoters to control each gRNA (Figure 1E).
A serial of studies have suggested that mismatches at the 50 This method was successfully demonstrated to enable multiple
end of spacer sequence are generally better tolerated than those editing (Jakociunas et al., 2015). For another strategy, the crRNA
at the 30 end, and especially the 8–12 bps at the 30 end of matrix and tracrRNA were expressed separately by RNAPIII
the spacer sequence are crucial for target recognition (Cong promoters, and processed into mature crRNA by RNase III and
et al., 2013; Fu et al., 2013; Hsu et al., 2013; Jiang et al., 2013; unknown nuclease(s) (Figure 1F), which also showed high gene
Sander and Joung, 2014). It is crucial to design gRNAs for disruption efficiency in S. cerevisiae (Bao et al., 2015). The tRNA-
CRISPR system, and a well-selected gRNA would minimize the processing system, which precisely cleaves both ends of the tRNA
risk of CRISPR-mediated DSBs at unwanted sites in genome precursor by RNase P and RNase Z (or RNase E in bacterium,
(off-target effects) and maximize the editing efficiency at the Figure 1G), exists in virtually all organisms and can be broadly
selected site (on-target activity) (Stovicek et al., 2017). Several used to boost the targeting capability and editing efficiency of
rules and algorithms have been proposed, and web-tools for CRISPR/Cas systems (Xie et al., 2015; Port and Bullock, 2016;
TABLE 1 | List of selected Web-sites for gRNA design in multi-species.
Name Link PAM Organism Function References
CHOPCHOP v3 http://chopchop.cbu.uib.no Most reported PAMs or Over 200 genomes Knock out/knock Labun et al., 2019
a self-defined sequence in/activation/repression/
Nanopore enrichment
E-CRISPR http://www.e-crisp.org/ Most reported PAMs Over 50 genomes Single design/paired Heigwer et al., 2014
designs
ATUM https://www.atum.bio/ NGG/NAG Homo sapiens/Mus
eCommerce/cas9/input musculus/Saccharomyces
cerevisiae/Escherichia
coli/Arabidopsis
thaliana
CRISPRdirect https://crispr.dbcls.jp/ Self-defined PAM Over 200 species Naito et al., 2015
CRISPR-ERA http://crisprera.stanford.edu/ NGG Human/mouse/rat/ Gene editing/ Liu et al., 2015
zebrafish/ activation/repression
D. melanogaster/
C. elegans/S. cerevisiae/
E. coli/B. subtilis
CC TOP https://crispr.cos.uni- Most reported PAMs 102 species gRNA and off-target Stemmer et al., 2015
heidelberg.de prediction
Qi et al., 2016; Ding et al., 2018; Zhang et al., 2019). Single or mutagenic pathway for DSB repair that occurs in whole cell
multiple gRNAs can be expressed by one promoter but separated cycle (Chang et al., 2017); whereas HDR is a slow, accurate,
by tRNA scaffolds [e.g., a 71 bp long pre-tRNAGly (Xie et al., 2015; template dependent pathway for both DSB and SSB repair,
Zhang et al., 2019)]. but only occurs in S/G2 phase (Ranjha et al., 2018). NHEJ
It is costly and time consuming for the sub-cloning of plasmids introduces unpredictable patterns of insertions and deletions, but
used for multi-loci editing, and some strategies could be taken if multiple DSBs are present, large deletions or chromosomal
for saving cloning time or improving editing efficiency. Gibson rearrangements may occur (Chang et al., 2017; Ranjha et al.,
assembly, Golden gate cloning and USER cloning have showed 2018). On the other hand, CRISPR/Cas mediated precise genome
high rates in multi DNA fragments assembly, which simplifies editing relies on DSB or SSB repairing through HDR pathway and
cloning steps for multiple gRNA expression cassettes, and thus DNA template (donor DNA).
saves the processing time (Bao et al., 2015; Shi et al., 2016; Smith Both single strand DNA (ssDNA) and double strand DNA
et al., 2016; Jensen et al., 2017; Zhang et al., 2019). Meanwhile, (dsDNA) fragments can be used as donors for genome editing.
in vivo homologous recombination has been reported for rapid Despite ssDNA donors showed higher editing efficiency than
assembling a certain plasmid backbone and PCR cassettes bearing dsDNA donors in several researches (Ran et al., 2013b; Miura
sgRNAs in some yeast species (S. cerevisiae and K. lactis), thus et al., 2015; Singh et al., 2015), dsDNA donors (linear or circular)
saving cloning steps for high-efficiency engineering (Horwitz showed comparable efficiency but higher flexibility and have
et al., 2015; Generoso et al., 2016; Reider Apel et al., 2017). been widely adopted for gene deleting, mutation and insertion
(DiCarlo et al., 2013; Zerbini et al., 2017; Zhang et al., 2019).
A DNA donor could be provided as HDR template to destroy
DNA Repair and Donor Design for DNA the target open reading frame, and change or eliminate gRNA
Deletion, Insert and Mutation binding sequence and PAM to avoid repeated cleavage by Cas
The CRISPR/Cas mediated precise genome editing relies on protein (Figures 2A,C; Raschmanova et al., 2018; Zhang et al.,
intrinsic DNA repair mechanisms after a DSB or SSB was 2019). A short dsDNA donor, with ∼50 bp homologous sequence
introduced to genome by a Cas protein, e.g., Cas9 nuclease or at each end, is usually viable and can be prepared by PCR of
a Cas9 mutant (Cas9 nickase, nCas9) (Figures 2A,B). There two oligonucleotide primers (Jakociunas et al., 2015; Zhang et al.,
are two main pathways for DSB repair in nearly all organisms: 2019). Such short donors can also be used for introduction of
non-homologous end-joining (NHEJ), direct ligation of two single-nucleotide mutations within different gene loci (Wang Y.
break ends with little or no sequence homology required; et al., 2016), if the locus to be edited is within the “GG” loci of a
and homology-directed repair (HDR), repairing DSB according PAM or the 20 nt protospacer. Long dsDNA donors can be used
to a DNA template with homology sequence (Figure 2A; for insertion (Figure 2D). Expression cassettes or other inserts
Ceccaldi et al., 2016; Ranjha et al., 2018). Despite alternative can be carried by long donors and inserted to genome through
end joining [alt-EJ, also termed microhomology-mediated end- HDR pathway (Figure 2D). These donors should have long
joining (MMEJ)] and single-strand annealing (SSA) may also homology arms (0.1–3 kb) for efficient HDR (Doetschman and
repair DSBs in some organisms, NHEJ and HDR remain Georgieva, 2017), and up to 24 kb fragments have been integrated
dominant pathways in most organisms (Ceccaldi et al., 2016; to yeast genome through CRISPR/Cas9 system (Shi et al., 2016,
Ranjha et al., 2018). NHEJ is a fast, template independent and 2019). Recently, transposons were proposed as an alternative
FIGURE 2 | DNA repair and donor design for DNA deletion, insert and mutation. (A) The Cas9-sgRNA complex binds to DNA target and triggers a double strand
break (DSB), which is subsequently repaired generally through non-homologous end joining (NHEJ) or homology-directed (HDR) pathway. NHEJ directs ligation of
two break ends with little or no sequence homology required, resulting in small insertions or deletions (indels); while HDR repairs DSB according to a DNA template
with homology sequence, resulting in precise editing when supplemented with ds- or ss-DNA donors. (B) A Cas9 nickase mutant with HNH or RuvC inactive domain
introduces a single strand break (SSB), which can be repaired by HDR rather than NHEJ pathway. (C) Donor designs for gene interruption, deletion and mutation.
Gene interruption: Small deletion (e.g., 8 bp) or insertion is integrated to shift reading frame, or stop codon is introduced to interrupt gene translation. Gene deletion:
A donor fused with sequence upstream and downstream ORF is sued for gene deletion (‘*’ indicate the deleted gene). Gene mutation: Sequence mutations can be
introduced by a donor, where seed sequence and PAM should be destroyed to avoid cutting again by Cas9-sgRNA complex. Chr, chromosome. (D) Donor design
for sequence insertion. A donor contain long sequence is integrated through HDR, and longer homology arms are required when inserting long sequence. (E)
Another strategy employing CRISPR I-F or V-K (e.g., Cas12k) mediates DNA integration with Tn7-like transposons (e.g., tnsB/tnsC/tniQ).
tool to mediate DNA integration via a HDR independent way microbial hosts (Freed et al., 2018; Raschmanova et al., 2018;
(Klompe et al., 2019; Strecker et al., 2019), which depends on type Palazzotto et al., 2019; Wang and Coleman, 2019; Ng et al., 2020),
I–F or V-K CRISPR effectors (e.g., Cas12k) and interacts with it is still challenging to construct CRISPR system with high
Tn7-like transposons (e.g., tnsB/tnsC/tniQ) (Figure 2E). editing efficiency, and/or apply various CRISPR strategies in
non-model microorganisms. In particular, lessons have also been
learned that several limitations should be overcome to enable the
Adaption of CRISPR/Cas System to multiplexed /genome-scale processing of CRISPR in non-model
Non-model Microorganisms microorganisms, such as the delivery of gRNAs or Cas proteins,
As a powerful toolbox for genome editing and regulation, the genotoxic stress, etc.
CRISPR systems are highly valued not only for model One dominant challenge is active, reliable and sufficient
microorganisms (e.g., E. coli, S. cerevisiae), but also provide more expression of Cas protein and gRNAs in a non-model host.
applicable perspectives for non-model microorganisms that are Due to the limited knowledge of non-conventional organisms,
difficult to be processed through traditional methods. Despite it is necessary to identify expression architectures ahead of
CRISPR/Cas systems have already been applied in plenty of CRISPR system construction. Constitutive or inducible RNAPII
promoters are used for expression of Cas proteins, but RNAP elsewhere on chromosome (Fu et al., 2013; Hsu et al., 2013;
III promoters should be used for sgRNA expression. In some O’Geen et al., 2015). The off-target effect may lead to unexpected
organisms without identified RNAPIII promoters, RNAPII DNA mutations, which limits the application of CRISPR in
promoters can also be used to express gRNAs when proper various organisms. Efforts to address this issue have been
strategies adopted when fusing sgRNA with special elements made to increase CRISPR specificity and to predict possible
at each end, e.g., Hammerhead ribozyme, HDV ribozyme, and off-target loci on genome. A well designed gRNA would largely
Csy4 cutting site (Jacobs et al., 2014; Nissim et al., 2014; Nowak reduce the crisis of off-target (Wang and Coleman, 2019),
et al., 2016; Weninger et al., 2016). Some architectures for and the “seed” sequence of gRNA (10–12 bp adjacent to the
stable episomal expression could also largely improve CRISPR PAM) highly decides the Cas9 cleavage specificity (Jinek et al.,
efficiency, such as centromeric sequence (Cao et al., 2017, 2020) 2012). To reduce the off-target risk and protect binding and
and autonomously replicating sequences (ARSs) (Gu et al., 2019). cleavage activity, bioinformatic tools or websites have been
Usually, the Cas9 form S. pyogenes (SpCas9) is efficient enough developed for gRNA design, such as Cas-OFFinder1 (Bae et al.,
for genome editing in different organisms. Codon optimization is 2014) and CCTop2 (Stemmer et al., 2015). Using truncated
occasionally needed when the wildtype SpCas9 was not actively sgRNAs (17-18 bp) showed reduced off-target effect with
expressed. In some organisms, however, SpCas9 showed low Cas9 nuclease and paired Cas9 nickases in human cells (Fu
efficiency or toxic effect, and repressed cell growth significantly et al., 2014). sgRNAs with two unpaired Gs on the 50 end also
(Ungerer and Pakrasi, 2016; Wendt et al., 2016; Jiang et al., 2017). showed more sensitive to mismatches in human cells (Kim
To solve this issue, different CRISPR systems or effector variants et al., 2015). Engineering of the Cas9 protein for fidelity or
(e.g., Cas12a) showed high editing efficiency but lower toxicity, specificity improvement also largely reduces off-target effects:
and were applied in those organisms (Ungerer and Pakrasi, 2016; e.g., Kleinstiver et al. (2016) reported a high-fidelity variant,
Jiang et al., 2017; Yeo et al., 2019). SpCas9-HF1 (N497A/R661A/Q695A/Q926A); Slaymaker et al.
On the other hand, the CRISPR aided precise, time-saving (2016) engineered several SpCas9 variants with high efficiency
and markerless genome editing relays on introducing DSBs at and specificity, e.g., eSpCas9(1.0) (K810A/K1003A/R1060A),
DNA targets and repairing process thereafter. Thus the intrinsic and eSpCas9(1.1) (K848A/K1003A/R1060A); Chen J. S. et al.
DNA repairing system largely determinates editing efficiency (2017) reported a new hyper-accurate Cas9 variant, HypaCas9
in non-model microorganisms. DSB repairing through NHEJ (N692A/M694A/Q695A/H698A), which demonstrated high
pathway results in small random deletions or inserts at the site genome-wide specificity without compromising on-target
of DSB, rather than precise repairing according to a template activity; Hu et al. (2018) reported an expanded PAM SpCas9
through HDR pathway. Thus, in those NHEJ dominant species, variant, xCas9 (xCas9-3.7: A262T, R324L, S409I, E480K, E543D,
CRISPR/Cas system can be used for just gene inactivation, but M694I, and E1219V), which showed much improved specificity
very low efficiency in precise DNA insertion, unless NHEJ is and more broad PAM sequence, e.g., ‘NG,’ ‘GAA,’ and ‘GAT.’ A
blocked, e.g., by knocking out KU70 and/or KU80 as mentioned Cas9 nickase mutant (nCas9) system can also reduce off-target
before (Gao S. et al., 2016; Schwartz et al., 2016; Cao et al., 2018; effect, in which a pair of guide RNAs is designed to bind to a
Bae et al., 2020). In some species lacking HDR pathway, phage- narrow target region and thus nCas9 complexes introduce two
derived recombinases (RecET and λ-Red) should be expressed SSBs on both strand of DNA, forming a DSB with sticky ends
to assist genome editing (Jiang et al., 2015; Wang B. et al., (Mali et al., 2013a; Ran et al., 2013a; Shen et al., 2014). Similarly,
2018). In addition, some chemical reagents can be supplemented Guilinger et al. fused catalytically inactive Cas9 (dCas9) and FokI
to increase HDR efficiency, such as SCR7 (Maruyama et al., nuclease (fCas9), which produces DSB by simultaneous binding
2015), RS-1(Song et al., 2016), KU0060648, and NU7441 (Robert of two fCas9 monomers to the DNA target sites ∼15 or 25 base
et al., 2015). The HDR pathway is the dominant mechanism pairs apart, and resulted in at least 4-fold higher specificity than
for DSB repair in most bacteria, and NHEJ is present in some that of paired nickases (Guilinger et al., 2014; Tsai et al., 2014;
bacteria including Mycobacterium, Pseudomonas, and Bacillus Wyvekens et al., 2015).
(Weller et al., 2002; Shuman and Glickman, 2007). In most Till now, the CRISPR/Cas system has already become the most
eukaryote, however, NHEJ is the dominant mechanism for DNA commonly used gene editing tool for numerous species. It has
repairing. It is recently reported that expression of T4 DNA become a precise, convenient and portable platform for genome
ligase provides efficient in vivo NHEJ repairing pathway in editing and beyond.
bacteria (Su et al., 2019). Donors also vary between organisms.
In some cases, short (∼50 bp) homologous arms (HAs) are
sufficient for HDR (Jakociunas et al., 2015; Zhang et al., REGULATION OF GENE EXPRESSION
2019); while in other cases, long (∼1–3 kb) HAs are preferred BY CRISPR/CAS TOOLBOX
(Doetschman and Georgieva, 2017).
In addition to site-specific gene editing, the catalytically dead
Efforts to Reduce Off-Target Effects Cas protein (e.g., dCas9, with H840A and D10A mutation)
Despite Cas9 cleavages DNA target depending on a 20 nt spacer that retained its capability to recognize and bind a target DNA
sequence of gRNA and PAM, it still potentially introduces
an undesired DSB at an unintended chromosomal locus (off- 1
http://www.rgenome.net/cas-offinder/
target), possibly because of gRNA binding to a similar sequence 2
https://crispr.cos.uni-heidelberg.de
sequence (Qi et al., 2013) has been developed as a multi- Bikard et al. (2013) reported a fusion protein between dCas9
functional platform based on its DNA recognizing and binding and the omega subunit (ω) of RNA Polymerase (dCas9-ω) that
properties. The CRISPR/dCas9 system has been intensively can activate transcription by binding at an optimal distance from
researched and applied for transcription regulation, complex the promoter in E. coli. However, this activation effect varied
metabolic engineering, directed revolution, gene target screening depending on the binding position and the innate promoter
and activation of silent gene clusters (Lino et al., 2018; Tarasava strength, with highest activation observed for weak promoters
et al., 2018; Xu and Oi, 2019). Especially, the CRISPR interference (Bikard et al., 2013). In S. cerevisiae, one commonly used
(CRISPRi) (Qi et al., 2013) and the CRISPR activation (CRISPRa) activator domain is VP64, consisting of four tandem copies
(Tanenbaum et al., 2014) that allow programmed controlling of of Herpes Simplex Viral Protein 16. dCas9-VP64 (Figure 3F)
gene expression without altering the genome, are effective tools increased target gene expression by 2.5-fold, and when multiple
for metabolic engineering, and are highlighted here. operators were targeted, the expression reached up to 70-fold
improvement (Farzadfard et al., 2013). Chavez et al. fused dCas9
with a tripartite activator VP64-p65-Rta (VPR, Figure 3G), which
Repression of Gene Expression by showed higher activating effect (∼10-fold) than dCas9-VP64
dCas9 (CRISPRi) counterparts (Chavez et al., 2015). Zalatan et al. (2015) tested
CRISPR interference (CRISPRi) represses expression of targeted “scaffold RNAs” (scRNA) that encode both target locus and
genes in a simple and reversible way without altered DNA MS2, PP7, or com RNA hairpins, recruiting their cognate RNA-
sequence or off-target effects (Qi et al., 2013). Especially binding proteins fusing with VP64 for transcriptional activation.
for those organisms lacking the RNA interference pathway, When the scRNA with two RNA hairpins connected by a
CRISPRi system offers an easy and efficient approach for double-stranded linker was used, stronger activation effects were
targeted gene knockdown (Li et al., 2016; Peters et al., observed (Zalatan et al., 2015). In a recent research, Dong et al.
2016). The CRISPR/dCas9 system was first used for repressing found that an activating effector, SoxS showed the highest effect
transcription by sterically hindering the RNA polymerase among E. coli regulators (SoxS, MarA, Rob, and CAP), Hijackers
recruiting (Figure 3A) or RNA polymerase processivity along the (TetD, λcII, GP33, and N4SSB ) and RNAP subunits (αNTD,
coding sequence (Figure 3B; Qi et al., 2013). Ni et al. (2019) RpoZ, and RpoD) in a CRISPRa system with gRNA scaffold MS2-
developed a CRISPRi method in which multi-gRNA plasmid was MCP interaction in E. coli (Dong et al., 2018). Especially, a SoxS
constructed that could down-regulate 7 genes simultaneously in mutant SoxSR93A and 5 aa linker further increased the activation
S. cerevisiae. However, this ‘road blocker’ strategy using dCas9 activity (Dong et al., 2018). Konermann et al. (2015) reported a
alone is not always efficient in some organisms (Qi et al., 2013). synergistic activation mediator (SAM) system for transcriptional
Gilbert et al. compared different repressive effector domains, activation (Figure 3H), which combined dCas9-VP64 with a
including the KRAB (Krüppel associated box) domain, the modified scRNA system. The activator domain of p65 and the
WRPW domain and the CS (Chromo Shadow) domain, and human heat shock factor 1 (HSF1) were fused with MS2 coat
found that dCas9-KRAB was the best repressor when targeting to protein (MCP), and bound to MS2 hairpins on sgRNA for
a window of -50 to +300 bp relative to the transcription start site transcription activation (Konermann et al., 2015). Tanenbaum
(TSS), or 0–100 bp region just downstream of the TSS (Figure 3C; et al. developed a dCas9-SunTag system with strong activation of
Gilbert et al., 2013, 2014). Another dCas9 fusion domain, Mxi1, a endogenous gene expression (Figure 3I), where the dCas9 was
mammalian transcriptional repressor domain that is reported to fused to a multimeric peptide (GCN4) array (SunTag), which
interact with the histone deacetylase Sin3 homolog in yeast, also can recruit multiple copies of scFv-VP64 for gene activation
showed effective repression in yeast (Gilbert et al., 2013; Jensen (Tanenbaum et al., 2014). Zhou et al. designed a new activation
et al., 2017; Schwartz et al., 2017; Geller et al., 2019; Wensing system, named as SunTag-p65-HSF1 (SPH), by combining the
et al., 2019). In another research, KRAB was fused to RNA- peptide array of SunTag and P65-HSF of SAM, which showed
binding domains (COM-KRAB) and achieved similar repression the highest level of activation compared to SAM, VPR, VP64 and
effects when targeting DNA sites overlapped the TSS using a SunTag in HEK293T and N2a cells (Zhou et al., 2018). Hilton et al.
scaffold RNA (scRNA) (Figure 3D; Zalatan et al., 2015). Kearns reported another strategy that fused dCas9 to the catalytic core of
et al. fused NmdCas9 with the histone demethylase LSD1, which the human acetyltransferase p300 (Figure 3J). This fusion protein
suppressed the expression of genes controlled by the targeted binds to upstream of a gene target, and catalyzes acetylation of
enhancers (Figure 3E; Kearns et al., 2015). histone H3 lysine 27 at its target sites, resulting in transcriptional
activation (Hilton et al., 2015).
Activation of Gene Expression by dCas9

(CRISPRa) Orthogonal CRISPR Systems for
When dCas9 is fused with transcriptional activator and binds to Comprehensive Engineering
the specific genomic locus, it can efficiently activate transcription In metabolic engineering and synthetic biology, complex
via recruitment of RNA polymerase (RNAP). This CRISPR engineering, e.g., overexpression, dynamic regulation, knock-
mediated transcriptional activation (CRISPRa) strategy has been down, and knock-out of multiple gene targets, is often required.
applied in both prokaryotic and eukaryotic cells, and several Unfortunately, such engineering processes are often carried
transcriptional activators have been reported. out sequentially and with low throughput. The development
FIGURE 3 | The nuclease-deactivated Cas9 (dCas9) mediated CRISPRi and CRISPRa system. (A) dCas9 blocks recruiting of RNA polymerase (RNAP). (B) dCas9
can sterically block the transcriptional elongation of RNAP. (C) dCas9 fuses repressors (e.g., KRAB, Mxi1) to repress gene transcription. (D) KRAB is fused to
RNA-binding domains (e.g., COM-KRAB) and achieves gene repression when targeting DNA sites overlaps the TSS using an scaffold RNA. (E) Fusion of dCas9 with
the histone demethylase LSD1 suppresses gene expression. (F) Fusion of dCas9 with activators (e.g., VP64, ω-subuint of RNAP) activates gene transcription. (G)
The VPR strategy for gene activation. The dCas9 has been fused to the combinatory transcriptional activator VP64-p65-Rta (VPR) to amplify the activation effects.
(H) The SAM system. The dCas9 is fused to VP64 and the sgRNA has been modified to contain two MS2 RNA aptamers to recruit the MS2 bacteriophage coat
protein (MCP), which was fused to the transcriptional activators p65 and heat shock factor 1 (HSF1). (I) The SunTag system. The tandem repeats of a small peptide
GCN4 are utilized to recruit multiple copies of scFv (single-chain variable fragment) in fusion with the transcriptional activator VP64. (J) dCas9 is fused with the
catalytic core of the human acetyltransferase p300, which catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional
activation.
of CRISPR toolbox enables nearly all engineering types, and and gene deletion (CRISPR-AID, Figure 4A) in the yeast
comprehensive applications of various CRISPR tools could solve S. cerevisiae. This orthogonal tri-functional CRISPR system
this problem. Vanegas et al. (2017) developed a CRISPR/CRISPRi employed dLbCpf1-VP for CRISPRa, dSpCas9-RD1152 for
system termed SWITCH, where the Cas9 cassette was integrated CRISPRi, and SaCas9 for CRISPRd (gene deletion), which
into genome for genetic engineering as stage 1; and then the recognize different type of sgRNA and PAMs (Lian et al., 2017).
dCas9 cassette was integrated and replaced the Cas9 cassette for By combining array-synthesized oligo pools, CRISPR-AID was
transcriptional regulation as stage 2 in S. cerevisiae. However, further developed as a genome-wide system (MAGIC) to generate
the SWITCH system does not enable genomic engineering diversified genomic libraries to identify genetic determinants of
and regulation control simultaneously. Lian et al. (2017) complex phenotypes in yeast (Lian et al., 2019). This system
developed an orthogonal tri-functional CRISPR system that was highlighted for complex engineering (gene interference,
combines transcriptional activation, transcriptional interference, activation and deletion), high coverage (nearly 100% ORFs
FIGURE 4 | Orthogonal CRISPR systems. (A) The orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and
gene deletion (CRISPR-AID). This orthogonal tri-functional CRISPR system employed dLbCpf1-VP for CRISPRa, dSpCas9-RD1152 for CRISPRi, and SaCas9 for
CRISPRd (gene deletion), which recognized different type of sgRNA and PAMs. dLbCpf1, dCpf1 from Lachnospiraceae bacterium ND2006; dSpCas9, dCas9 from
S. pyogenes; SaCas9, Cas9 from S. aureus. (B) The orthogonal CRISPR system with different RNA scaffolds. The gRNA fused with MS2 is used for activation
through binding of MCP-VP64 or MCP-SoxSR93A . The gRNA fused with com is used for repression through binding of Com-KRAB. Alternatively, a sgRNA without
MS2 or com scaffold can hinder gene expression either. (C) CRISPR and CRISPRi via different crRNA length. Cas12a triggers DSB and genome editing with
20 bp-spacer in crRNA, while it blocks transcription with a short crRNA (16 bp-spacer).
and RNA genes) and iterative/simultaneous construction, which was used for both gene editing and repression simultaneously
enabled identification of new gene targets and interactions by supplemented crRNA with different length (Figure 4C),
for furfural tolerance as a demonstration (Lian et al., 2019). where a 20 bp-crRNA triggers DSB and genome editing, but a
Combining orthogonal CRISPR and CRISPRi enables genome 16 bp-crRNA results in gene repression without DNA cleavage
engineering and transcriptional regulation in E. coli, where (Liu W. et al., 2019).
orthogonal Cas protein candidates were expressed for CRISPR CRISPR system can also be dynamically controlled by
and CRISPRi separately and simultaneously (Sung et al., 2019). chemical or light with specific wavelength (ligand). Generally,
Sung et al. (2019) harnessed the St1Cas9 (from Streptococcus a ligand induces dimerizing of two ligand binding domains
thermophilus) for DNA cleavage and insertion, and the SpdCas9 (LBDs), and each domain can be fused to dCas9 and transcription
for CRISPRi. In addition to orthogonal effectors, RNA scaffold effector (e.g., VPR for activation, and KRAB for repression),
and binding protein can also be used for CRISPRi and CRISPRa respectively. In such a ligand inducible CRISPRa/CRISPRi
simultaneously. Zalatan et al. used “scaffold RNAs” (scRNA) to system, the presence of ligand will induce the binding of dCas9
recruit activators or repressors (e.g., using MS2 to recruit MCP- and effector, and thus activate or repress the downstream gene
VP64 and com to recruit Com-KRAB, Figure 4B; Zalatan et al., expression. Several ligands have been reported for development
2015). Thus, genes are activated or repressed depending on the of inducible CRISPR systems, including abscisic acid (inducing
scRNA features instead of Cas9 orthologs. Another strategy of dimerization of ABI-PYL1) (Gao Y. et al., 2016; Bao et al., 2017;
simultaneous activation and interference was achieved by using Chen T. et al., 2017), gibberellin (inducing dimerization of GID1-
one dCas9 protein but MS2 scRNAs for activation by recruiting GAI24) (Gao Y. et al., 2016), rapamycin (inducing dimerization
MCP-(5aa)-SoxSR93A , while an unmodified gRNAs for repression of FKBP–FRB) (Zetsche et al., 2015b; Bao et al., 2017), magnet
(Figure 4B; Dong et al., 2018). On the other hand, one Cas12a (inducing dimerization of pMag–nMag) (Nihongaki et al.,
2015a,b, 2017; Polstein and Gersbach, 2015), blue light (inducing Another method for processing precise base editing is to use
dimerization of CRY2-CIB1), and phytochrome-based red light dCas9 fused deaminase, which hydrolyzes the amine group of ‘C’
(inducing dimerization of PhyB–PIF (Levskaya et al., 2009). and ‘A,’ and enables ‘C’ to ‘T’ and ‘A’ to ‘G’ conversions without
When orthogonal dCas proteins are used to response to different dsDNA cleavage (Figures 5B,C). Cytidine base editors (CBEs)
ligands and effector-LBDs, the CRISPR system is expected for and adenine base editors (ABEs) were developed to convert
complex, dynamic, and programmable regulations (Gao Y. et al., ‘C’ to ‘T’ (Komor et al., 2016) and ‘A’ to ‘G’ (Gaudelli et al.,
2016; Bao et al., 2017; Hill et al., 2018; Xu and Oi, 2019). 2017) separately. Typically, BE3 (the mainly used CBE, cytidine
deaminase-nCas9-UGI) and ABE7.10 (the most widely used
Precise Single Base Editing With CRISPR ABE, wtTadA-mutantTadA-nCas9) showed the highest editing
Since Cas9 can tolerate mismatches in the 20 bp gRNA binding efficiency within the protospacer position 4–8 and 4–7 (counting
region, single-nucleotide mutations in this region could be bound the PAM as positions 21–23) (Komor et al., 2016; Gaudelli et al.,
and cleaved again. Thus, single-nucleotide mutations become 2017). CBEs using LbCpf1 showed an editing window preference
difficult for CRISPR system. Such repeated cleavage can be of positions 10-12 (Li et al., 2018). In a recent research, a single-
avoided by introduction of additional mutations to eliminate base editing termed CRISPR-BEST was developed by fusing Cas9
the gRNA target site or the PAM sequence (DiCarlo et al., nickase (D10A) to cytidine and adenosine deaminase as editors.
2013; Jakociunas et al., 2015; Laughery et al., 2015). However, The CRISPR-BEST enabled ‘C→T’ and ‘A→G’ conversion within
extra mutations are introduced for avoiding repeated cleavage. a window of approximately 7 and 6 nucleotides, respectively,
A two-step strategy (Figure 5A) was developed for precise single with high efficiency in Streptomyces species (Tong et al., 2019).
mutation by introducing the CRISPR/Cas9 twice (Biot-Pelletier In another research, Zhao et al. fused dCas9 with PmCDA1
and Martin, 2016; Paquet et al., 2016; Wang Y. et al., 2016). (the cytidine deaminase from Petromyzon marinus) and UGI
In the first step, the target was eliminated by insertion of a (the uracil DNA glycosylase inhibitor), which enabled point
20 nucleotide heterologous stuffer sequence via CRISPR/Cas9 mutations from ‘C’ to ‘T’ (‘C→T’) in Streptomyces coelicolor,
system; and in the second step, this stuffer was eliminated by the and the efficiency reached up to 100%, 60%, and 20% for one,
original sequence with desired point mutation via CRISPR/Cas9 two and three loci, respectively (Zhao et al., 2019). Wang et al.
system (Biot-Pelletier and Martin, 2016; Paquet et al., 2016; fused Cas9 nickase (D10A) with activation-induced cytidine
Wang Y. et al., 2016). deaminase, which enabled precise ‘C→T’ conversion at one,
FIGURE 5 | Strategies for precise single base editing. (A) Two-step stuffer-assisted point mutation. In the first step, a 20-nucleotide target genome sequence close
to the target is replaced by a heterologous stuffer fragment via homologous recombination. In the second step, the stuffer fragment acts as the target sequence,
recognized by a second gRNA, and the original sequence with arbitrary mutation is inserted back. Chr, chromosome. (B) ‘C→T’ mutation through DSB independent
pathway. The dCas9 or Cas9 nickase (nCas9, D10A) is fused with cytidine deaminase and uracil DNA glycosylase inhibitor (UGI), and binds to a DNA target. Cytidine
deaminase converts the cytidine (‘C’) to uracil (‘U’) in the non-targeted strand, which is protected by UGI from the nucleotide excision repair (NER) pathway. And in
the next replication cycle, the ‘G:C’ base pair is repaired to ‘T:A’. (C) ‘A→G’ mutation through DSB independent pathway. The dCas9 or nCas9 is fused with
adenosine deaminase and binds to a DNA target. Adenosine deaminase converts the adenosine (‘A’) to hypoxanthine (‘I’) in the non-targeted strand. And in the next
replication cycle, the ‘T:A’ base pair is repaired to ‘C:G’.
two and three loci with an efficiency of 100%, 87%, and 23%, multi-loci editing because of the high HDR rate. Several multi-
respectively, in Corynebacterium glutamicum, and built a library loci editing systems have been developed, including CRISPRm,
of 14154 unique gRNAs for inactivation of 2726 genes (Wang HI-CRISPR, CasEMBLR, GTR-CRISPR (Ryan and Cate, 2014;
Y. et al., 2018). The low efficiency of triple-site editing could Bao et al., 2015; Jakociunas et al., 2015, 2018a; Zhang et al.,
be possibly caused by the lower amount of the base editor at 2019). Bao et al. expressed crRNA and tracrRNA separately, and
each locus than those targeting single loci. And a developed CAN1, ADE2 and LYP1 were simultaneously disrupted in 4 days
system with expanding targeting scope, editing window, and base with an efficiency ranging from 27 to 87%. Furthermore, another
transition capability was further constructed in C. glutamicum by three genes were simultaneously disrupted in 6 days with 100%
the same group (Wang et al., 2019). efficiency (Bao et al., 2015). Ryan and Cate developed a CRISPRm
system, where 1–3 sgRNAs were expressed by a tRNA promoter
and fused to the 30 end of the self-cleaving HDV ribozyme
APPLICATION OF CRISPR/CAS SYSTEM for protecting the sgRNA from 50 -exonucleolytic activities, and
achieved modifications of 1–3 targets with 81–100% efficiency
IN MICROBIAL BIOTECHNOLOGY (Ryan and Cate, 2014; Ryan et al., 2014). In another research,
sgRNAs were separated by a 28 nt stem-loop sequence and
The fast developed and multiple functioned CRISPR system
cleaved by Csy4 (a bacterial endoribonuclease from Pseudomonas
enables versatile, systematic and automatic applications in
aeruginosa) to generate multiple gRNAs from a single transcript
microbial technology. Especially, the CRISPR/Cas9 system has
for multiple gene deletion in S. cerevisiae (Ferreira et al., 2018).
been developed for fast, efficient, precise and concise multi-loci
This strategy enabled a deletion of 4 genes simultaneously with
editing and metabolic engineering. These researches imploring
an efficiency of 96% (Ferreira et al., 2018). Jakociunas et al. (2015)
CRISPR/Cas system for hyper or wider applications in recent
developed a strategy, termed CasEMBLR, for in vivo assembly
two years are shown in Table 2. And efforts for promoting
of gene cassettes and integrated to genome at up to 3 cleavage
CRISPR system for multi-loci editing and metabolic engineering
loci by CRISPR with high efficiency (30.6%, when optimized
are highlighted.
gRNAs were used). By using this method, 15 exogenous DNA
parts were correctly assembled and integrated into 3 genomic
Promotion of CRISPR/Cas System for loci for carotenoid production in one transformation (Jakociunas
Multi-Loci Editing et al., 2015, 2018a). Kildegaard adopted similar strategy for multi-
One bias of CRISPR/Cas system is that the Cas/sgRNA architecture assembly and insertion (Kildegaard et al., 2019).
complex can bind to more than one loci when proper sgRNAs Kuivanen et al. (2018) reported a high-throughput workflow for
are provided, which enables multi-loci editing simultaneously. CRISPR/Cas9 mediated combinatorial promoter replacements,
Several groups have developed the CRISPR/Cas9 system for more and successfully edited 3 loci simultaneously with a frequency of
efficient multi-loci editing, which makes genomic engineering 50%. Mans et al. used in vitro assembly for gRNAs expression
more efficient, simple and convenient. E. coli and S. cerevisiae are and achieved simultaneous deletion of up to 6 genes in a
typical model strains for prokaryotic and eukaryotic organisms, single transformation step with a high efficiency at 65% (Mans
respectively, and multi-loci editing strategies are well illustrated et al., 2015). Zhang et al. report a gRNA-tRNA array for
thereby, enlightening adapted multi-loci editing strategies in CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering, and
other organisms (Gao S. et al., 2016; Wang J. et al., 2018; Zhang simultaneously disrupted 8 genes with 87% efficiency, where
et al., 2018; Liu D. et al., 2019; Schultz et al., 2019; Tran et al., gRNAs were fused with tRNAGLY scaffolds and expressed in 2
2019; Zheng et al., 2019; Yang et al., 2020). quadruple arrays (Zhang et al., 2019). Besides, Zhang et al. also
E. coli is the most intensively researched prokaryotic model reported an accelerated Lightning GTR-CRISPR strategy, which
microorganism, and multi-loci editing mediated by CRISPR/Cas saving the cloning step in E. coli by directly transforming the
is typical in E. coli. Jiang et al. expressed SpCas9 and λ-Red in Golden Gate reaction mix (the successfully assembled plasmid
E. coli, and achieved 3 genes disruption at an efficiency of 47% contained sgRNA expression cassettes and a Cas9 expression
(Jiang et al., 2015). Ronda et al. expressed tracrRNA and crRNA cassette) to yeast (Zhang et al., 2019). Bao et al. developed a
separately, and achieved 2 genes disruption at an efficiency higher CRISPR-Cas9- and homology-directed-repair-assisted genome-
than 70% in E. coli (Ronda et al., 2016). Bassalo et al. (2016) scale engineering method named CHAnGE, to construct genetic
developed a rapid and efficient one-step engineering method, variant libraries in yeast (Bao et al., 2018). In CHAnGE, guide
and engineered 7 targets simultaneously with efficiencies ranging sequence and the homologous recombination (HDR) template
from 70 to 100%. Ao et al. (2018) expressed Cas12a instead were arranged and synthesized in a single oligonucleotide, and
of Cas9, resulting in the efficiency of integration of 2 loci at a oligonucleotide library of 24,765 unique guide sequences
40%, and the efficiency of integration of 3 loci at 20%. Sung targeting 6,459 ORFs was synthesized on a chip and then
et al. developed a method that combined orthogonal CRISPR assembled into a vector [pCRCT, harboring iCas9, tracrRNA
and CRISPRi and enabled constitutive knockdown of three genes, expression cassettes and a promoter for sgRNA expression, as
knock-in of pyc and knockout of adhE, without compromising reported in HI-CRISPR system (Bao et al., 2015)] to build a
the CRISPRi knockdown efficiency (Sung et al., 2019). pool of plasmids. This plasmid pool was then used to create a
Saccharomyces cerevisiae is the most intensively researched genome-wide gene disruption collection, in which more than
eukaryotic model microorganism, which enables highly efficient 98% of target sequences were efficiently edited with an average
TABLE 2 | Selected recent CRISPR mediated metabolic engineering works.
Host Toolbox Product Engineering by Achievements References

CRISPR
Bacillus subtilis CRISPRi Hyaluronic acid (HA) Reduce the expression Increased HA titer of up Westbrook et al., 2018
of pfkA or zwf to 108% at 2.26 g/L and
enhanced molecular
weight
Bacillus subtilis Xylose-induced CRISPRi N-acetylglucosamine Reduced the expression 103.1 g/L in fed-batch Wu et al., 2018b
of zwf, pfkA, glmM fermentation
Clostridium ljungdahlii CRISPR/Cas9 Butyric acid A butyric acid production 1.01 g/L of butyric acid Huang et al., 2019
pathway was integrated within 3 days by
fermenting synthesis gas
(CO2 /CO)
Clostridium ljungdahlii CRISPRi 3-Hydroxybutyrate (3HB) Repression of pta and Downregulation of pta Woolston et al., 2018
aor2 increases 3HB
production 2.3-fold with
a titer at 21 mM
Clostridium Type I-B CRISPR-Cas n-Butanol Deletion of spo0A and 26.2g/L Zhang et al., 2018
tyrobutyricum pyrF, and integration of
adhE1 or adhE2 to
replace cat1
Corynebacterium Cas9 nickase (D10A) Glutamate Construction of a Increased production by Wang Y. et al., 2018
glutamicum with activation-induced combinatorial gene 3-fold
cytidine deaminase inactivation library, and
pyk/ldhA double
inactivation for glutamate
production
Synechocystis sp. PCC Inducible CRISPRi n-Butanol Repression of gltA 5-fold increase of carbon Shabestary et al., 2018
6803 partitioning to n-butanol
relative to a
non-repression strain
Escherichia coli CRISPR-Cas12a 5-Aminolevulinic acid Integrating the T7 RNAP 1.55 g/L Ao et al., 2018
cassette and pT7-hem1
cassette into the lacZ
site and the torS site,
respectively
Escherichia coli Orthogonal CRISPR and Succinate Knock in pyc, knockout Increased by 178% with Sung et al., 2019
CRISPRi systems adhE and knockdown of a titer at 2.5 g/L, and the
ptsG, ldhA, and pflB titer increased to 15 g/L
in a fermenter
Escherichia coli CRISPRi Naringenin 7-sulfate Increased bioconversion Chu et al., 2018
rate by 2.83-fold
(48.67%)
Escherichia coli Iterative CRISPR 3HP 13 rounds of editing Increased by up to Liu et al., 2018a,b
EnAbled Trackable using iCREATE 60-fold with a titer at
genome Engineering 30 g/L
(iCREATE)
Escherichia coli PS-Brick assembly and 1-Propanol ppc, aspA, aspC, asd, 1.35 g/L in fed-batch Liu S. et al., 2019
CRISPR/Cas9 pntAB, thrA443 BC, rhtC fermentation
were overexpressed and
tdh and ilvA were
deleted for threonine
production; kivD and
ADH2 were expressed in
A443 BC and asd
expressed strain for
1-propanol production.
Escherichia coli CRISPR/Cas9 Uridine Expression of pyrimidine 70.3 g/L in fed-batch Wu et al., 2018a
operon of Bacillus fermentation
subtilis and prs, and
deletion of lacI, rihC,
argF, thrA, iclR, purr,
nupC and nupG
(Continued)
TABLE 2 | Continued
Host Toolbox Product Engineering by Achievements References

CRISPR
Escherichia coli CRISPR/Cas9 Octanoic acid Overexpression of fabZ Increased by 61% with a Tan Z. et al., 2018
and deletion of fadE, titer at 442 mg/L and
fumAC and ackA further optimized to
1 g/L in fed-batch
fermentation
Escherichia coli CRISPR/Cas9 Itaconic acid Deletion of ldhA, poxB 3.06 g/L Yang et al., 2018
and pflB
Escherichia coli CRISPRi Isopentenol Reduced expression of Increased by 98% Tian et al., 2019
asnA, prpE and gldA
Escherichia coli CRISPR/Cas9 Uridine 5.6 g/L Li Y. et al., 2019
Halomonas CRISPR/Cas9 Poly(3-hydroxybutyrate- Deletion of sdhE and icl 6.3 g/L cell dry weight Chen et al., 2019
bluephagenesis co-3-hydroxyvalerate) (CDW), 65% PHBV in
(PHBV) CDW and 25mol% 3HV
in PHBV
Halomonas spp. CRISPR/Cas9 P(3HB-co-3HV) Deletion of prpC Increased 3HV fraction in Qin et al., 2018
consisting of the copolymers by
3-hydroxybutyrate (3HB) approximately 16-folds
and 3-hydroxyvalerate with a fraction at
(3HV) 11.81 mol%
Klebsiella pneumoniae CRISPRi 3HP Deletion of pmd, ldhA, Increased 3HP titer by Wang J. et al., 2018
aldA and mgsA 37% by reducing lactic
acid synthesis, and
further enhanced to
36.7 g/L 3-HP in
fed-batch cultivation
Saccharomyces GTR-CRISPR Fatty acids Knocking out of FAA1, Increased free fatty acids Zhang et al., 2019
cerevisiae FAA4, POX1, ARE2, by 30-fold with a titer at
PAH1, LPP1, DPP1, and 559.52 mg/L, and
ARE1 increased total fatty
acids by 1.8-fold with a
titer at 943.92 mg/L
Saccharomyces CRISPRi β-amyrin down-regulating ADH1, 156.7 mg/L Ni et al., 2019
cerevisiae ADH4, ADH5, ADH6,
CIT2, MLS2, and ERG7
Saccharomyces CRISPRa CRISPR/Cas-based Developed a Li P. et al., 2019
cerevisiae gene activation library CRISPR/Cas-based
gene activation library,
and improved
thermotolerance
Saccharomyces CRISPR mediated improved Mitsui et al., 2019
cerevisiae genome shuffling thermotolerance
Saccharomyces CRISPRi and in vivo cis, cis-Muconic acid Integration of multiple Increased the titer by Kildegaard et al., 2019
cerevisiae assembly expression cassettes 5–21%
and down-regulating of
ZWF1
Saccharomyces CRISPRi, construction of 2,3-Butanediol (BDO) Knocking down Increased BDO titer by Deaner et al., 2018
cerevisiae tRNA-sgRNA operons ADH1/3/5 and GPD1, 2-fold
using LEGO and overexpression of
BDH1
Saccharomyces CRISPRa 3HP A gRNA library targeting increased by 15 - 36% Ferreira et al., 2019
cerevisiae 168 genes
Synechocystis sp. CRISPRi Fatty alcohols Repression of aar, ado, Increased by 3-fold with Kaczmarzyk et al., 2018
sll1848, sll1752, slr2060, a specific titer of
and slr1510 octadocanol at
10.3 mg/g DCW
Ustilago maydis CRISPR/Cas9 Itaconic acid 1cyp3, 1MEL, 1UA, Increased by 10.2-fold Becker et al., 2019
and 1Pria1 ::Petef with a yield at 19.4 g/L
and further enhanced to
53.5 g/L under
optimized medium
frequency of 82% (Bao et al., 2018). In parallel, Jakociunas et al. Application of orthogonal CRISPR systems would also make
employed error-prone PCR to generate DNA mutant libraries as complex metabolic engineering work simpler and more efficient,
donor, and used Cas9-mediated genome integration to introduce and knocking-in, knocking-out, interference and activation could
mutations at single- or multi-loci with efficiencies reaching 98- be simultaneously processed for multiplex target genes. Several
99%, for robust directed evolution (Jakociunas et al., 2018b). excellent examples for orthogonal CRISPR aided metabolic
Besides, large chromosomal fragment deletion methods were engineering were demonstrated recently (Table 2). For example,
developed based on CRISPR/Cas9 system. Easmin developed Sung et al. employed a Cas9 protein from Streptococcus
a guide RNA-transient expression system (gRNA-TES), where thermophilus CRISPR1 (St1Cas9) to deliver DNA cleavage, and
two sgRNA expression fragments (locating to each end of target used the common dSpCas9 for gene interference (Sung et al.,
region on genome) and DNA donor containing CgLEU2 were co- 2019). Each Cas9 recognized its cognate sgRNA, and worked
transformed into host for a replacement of up to 500-kb regions orthogonally. Thus, St1Cas9 was harnessed to integrate SpdCas9
with efficiencies of 67-100% (Easmin et al., 2019). and sgRNA arrays, as well as knock in pyc and knockout
In multi-loci editing using CRISPR systems, co-expression adhE; whereas SpdCas9 was applied for constitutive knockdown
of many sgRNAs often requires repetitive DNA sequences of ptsG, ldhA, and pflB to eliminate competing pathways for
(e.g., repeated promoters/terminators and guide RNA scaffolds), lactate, formate, and ethanol synthesis. The final engineered
which possibly triggers genetic instability and phenotype loss. strain produced 2.5 g/L succinate with 178% improvement
Reis et al. reported a non-repetitive extra-long sgRNA arrays (Sung et al., 2019).
(ELSAs) strategy, where different promoters, terminators and the
sgRNAs’ 61-nucleotide handle sequences were characterized for
multiplex sgRNA expression (Reis et al., 2019). Through ELSAs, Other CRISPR Applications
22 sgRNAs within non-repetitive extra-long sgRNA arrays are The fast development of CRISPR tools enable various
simultaneously expressed for CRISPRi system, and repressed up applications beyond genome editing and transcriptional
to 13 genes by up to 3,500-fold in E. coli (Reis et al., 2019). The regulation. One application is building activated and/or
design of ELSAs and the identified 28 sgRNA handles that bind interfered gene libraries to screen phenotype related genes.
Cas9 can be adopted for CRISPR mediated multi-loci editing Gilbert et al. applied genomic libraries of CRISPRi and
for metabolic engineering and synthetic biology applications in CRISPRa to screen gene targets related to the sensitivity
other organisms. to a cholera-diphtheria toxin (Gilbert et al., 2014). Li et al.
build a CRISPR/Cas-based gene activation library, and used
it to screen gene targets for improved thermotolerance in
The CRISPR/Cas Mediated Metabolic S. cerevisiae (Li P. et al., 2019). Lee et al. used a CRISPRi
Engineering system, targeting 4,565 (99.7%) genes to identify a minimal
The developing powerful CRISPR toolbox enables advanced set of genes required for rapid growth of Vibrio natriegens
genome editing and transcription regulation, and has become the (Lee et al., 2019). Bassalo et al. applied CRISPR/Cas9 to
ideal strategy for metabolic engineering, because of its advantage perform a parallel and high-resolution interrogation of over
of ease of use, modularity, and scalability. Metabolic engineering 16,000 mutations to identify proteins associated to lysine
rewrites the metabolic network through single or multiple gene metabolism in E. coli (Bassalo et al., 2018). While Wang et al.
manipulation, to create or improve microbial cell factories for built a larger guide RNA library of ∼60,000 members for
the production of fuels, chemicals, pharmaceutics, etc. CRISPR coding and non-coding targets in E. coli, and applied CRISPRi
systems have been increasingly used in metabolic engineering system to associate genes with phenotypes at the genome level
field for construction of microbial cell factories (Yan and Fong, (Wang T. et al., 2018).
2017; Mougiakos et al., 2018; Tarasava et al., 2018), and those CRISPR system can also be used to discover novel compounds
recent works are summarized in Table 2. by activating the expression of silent gene or gene cluster, which
One advantage of CRISPR/Cas system is that it realizes precise may code enzymes for novel or undetectable nature products
genome editing at multi-loci in one transformation, without synthesis. Zhang et al. reported an one-step CRISPR/Cas9
integrating a marker gene on genome for selection, and thus it knock-in strategy to activate biosynthetic gene cluster expression
would largely simplify operation steps and save time and labor and trigger metabolite production by insertion of strong
in metabolic engineering works. As a proof of concept, Zhang promoters upstream biosynthetic operons in Streptomyces
et al. employed GTR-CRISPR to engineer lipid metabolism in species (Zhang et al., 2017; Lim et al., 2018). Grijseels et al.
S. cerevisiae for free fatty acid (FFA) production (Zhang et al., (2018) implemented the CRISPR/Cas9 technology to identify
2019). 8 genes in lipid metabolism were deleted through two the decumbenone biosynthetic gene cluster in Penicillium
rounds operation: FAA1, FAA4, POX1, and ARE2 were deleted decumbens, and evaluated the importance of targets for
in the first round; and after losing the plasmid through anti- production of calbistrin. Similarly, Lee et al. (2018) adopted
selection on 5-FOA medium, PAH1, LPP1, DPP1, and ARE1 the CRISPRi system for rapid identification of unknown
were knocked out in the second round transformation (Zhang carboxyl esterase activity in C. glutamicum. Naseri et al. (2019)
et al., 2019). Thus, the final strain with 8-gene deletion was employed orthogonal, plant-derived artificial transcription
constructed in 10 days, which produced 559.52 mg/L FFA with factors (ATFs) for the balanced expression of multiple genes
30-fold increase compared with wildtype. in S. cerevisiae, and generated CRISPR/Cas9-mediated cell
libraries for producing β-carotene and co-producing β-ionone predict and design efficient gRNAs for CRISPRa and CRISPRi
and biosensor-responsive naringenin. at single nucleotide level. Another highlighted direction is a
CRISPR/Cas9 system also amplified the power of evolutionary comprehensive application of different CRISPR/Cas systems
engineering for industrial microorganisms. Mitsui et al. to facilitate insertion, deletion and transcriptional regulation
developed CRISPR/Cas9 system as a genome shuffling method simultaneously. Lian et al. developed a such strategy in
for evolutionary engineering to obtain a thermotolerant mutant S. cerevisiae (Lian et al., 2017), enabling perturbation of the
strain (Mitsui et al., 2019). Halperin et al. (2018) proposed a metabolic and regulatory networks in a modular, parallel,
new method called EvolvR that can accelerate mutagenesis up and high-throughput manner, which is worthy to adapt such
to 7,770,000-fold within a tunable window length via CRISPR- strategy in other organisms. Besides, with the genome wide
guided nickases. Jakoèiûnas et al. reported a method named application of CRISPR and array-synthesized oligo pools, it
Cas9-mediated Protein Evolution Reaction (CasPER) for efficient is more easier to generate large libraries containing millions
mutagenesis of nucleotides by combining error-prone PCR and and even billions of variants (Lian et al., 2019). Therefore,
Cas9-mediated genome integration (Jakociunas et al., 2018b). developing high throughput techniques, e.g., high efficient
Garst et al. (2017) constructed CRISPR-enabled trackable genome transformation methods, robotic platforms and microfluidic
engineering (CREATE) method, where a library of targets was systems remain necessary and challenging. Furthermore, with
built and transformed for multiplex editing in vivo, followed the aid of automated robotic systems (HamediRad et al.,
by screening and mutation identification. Through CREATE, a 2019), CRISPR system could become more powerful for
library of 104 –106 individual members was built, and an average functional mapping and multiplex optimization of strains in an
mutation rate of 75% was reached for site saturation mutagenesis unprecedented scale.
for protein engineering and adaptive laboratory evolution (Garst On the other hand, types VI and III CRISPR systems were
et al., 2017). Based on CREATE, Liu et al. developed an iterative reported to have specialized or pluralistic for RNA targeting
CRISPR EnAbled Trackable genome Engineering (iCREATE) activity (Shmakov et al., 2017), which enabled direct RNA
strategy for the rapid construction of combinatorially modified engineering by CRISPR systems (Abudayyeh et al., 2016, 2017).
genomes, and used it for 3-hydroxypropionate (3HP) production Despite limitations in those RNA-targeting CRISPR systems
improvement (Liu et al., 2018a,b). [reviewed in Smargon et al. (2020)], it has showed capabilities
in RNA imaging (Abudayyeh et al., 2017), RNA interference
(Abudayyeh et al., 2016), RNA mutation (Abudayyeh et al., 2017)
CONCLUSION AND FUTURE and RNA detection (Gootenberg et al., 2017, 2018). Thus CRISPR
PERSPECTIVES aided RNA manipulation shows bright prospect as an emerging
tool in fundamental research and bioengineering.
The intrinsic advantage of CRISPR enables an evolutionary
and versatile platform for genotypic, metabolic and phenotypic
engineering in microbial biotechnology. The CRISPR based AUTHOR CONTRIBUTIONS
tools are generally with higher efficiency, more convenience,
more efficient multiplex targets editing/regulation and time- WD and SS outlined this manuscript. WD drafted the
saving compared with traditional ones. However, challenges manuscript. SS and YZ revised the manuscript. All authors
and weaknesses still exist. Despite the CRISPR/Cas system contributed to the article and approved the submitted version.
has been used for a broad range of microorganisms, the
genome editing efficiency varies between species to species and
even between cell to cell, indicating cellular intrinsic process
FUNDING
impacts CRISPR/Cas system. More reliable, inducible and widely This work was supported by the National Key Research
applicable expression architectures, e.g., RNAPII- and RNAPIII- and Development Program of China (2018YFA0901800 and
promoters, ARSs and centromere sequences can be developed for 2018YFA0900100), the National Natural Science Foundation of
multi-hosts, which would enable the expression of Cas effectors China (21878013), the China Postdoctoral Science Foundation
and gRNAs in different organisms with simple modification, (2019M650450), the Fundamental Research Funds for the
especially in non-model microorganism, which would make Central Universities (ZY1933), the Foundation of Key
CRISPR a portable platform and transplant CRISPR strategies Laboratory of Biomass Chemical Engineering of Ministry
from model microorganism to those non-model ones. The of Education, Zhejiang University (No. 2018BCE004), the
efficiency of CRISPR system (both for genome editing and Fundamental Research Funds for the Central Universities, and
transcriptional regulation tools) showed a gRNA position reliable the Beijing Advanced Innovation Center for Soft Matter Science
phenomenon, which means high-efficiency on some gRNAs, and Engineering.
but low-efficiency or even non-work on others. Therefore,
more than one gRNAs should be tested when editing a new
target, especially for efficient CRISPRa and CRISPRi. Thus, ACKNOWLEDGMENTS
it remains important for developing more powerful effectors
for robust activation/inference, engineering the Cas protein We thank Prof. Huimin Zhao (University of Illinois at Urbana-
for better performance, and developing algorithms that can Champaign) for his very enlightening comments.
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Yang, Z., Wang, H., Wang, Y., Ren, Y., and Wei, D. (2018). Manufacturing in this journal is cited, in accordance with accepted academic practice. No use,
multienzymatic complex reactors in vivo by self-assembly to improve the distribution or reproduction is permitted which does not comply with these terms.

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Development and Application of

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated

TABLE 1 | List of selected Web-sites for gRNA design in multi-species.

Name Link PAM Organism Function References

Activation of Gene Expression by dCas9

TABLE 2 | Selected recent CRISPR mediated metabolic engineering works.

Host Toolbox Product Engineering by Achievements References

Host Toolbox Product Engineering by Achievements References

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