Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis

doi:10.21203/rs.3.rs-1660810/v1

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Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis

https://doi.org/10.21203/rs.3.rs-1660810/v1

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Background: The true diploid frog, Xenopus tropicalis (X. tropicalis) is an excellent genetic model organism. To date, the CRISPR/Cas-mediated genome editing methods established in this species are mostly based on SpCas9 that requires the stringent NGG protospacer-adjacent motif (PAM) for target recognition, which limits its genome editing scope. Thus, it is highly desirable to circumvent this limitation.

Results: Through one-cell stage injection of Cas/gRNAs into X. tropicalis embryos, we evaluated the mutagenic efficiency of 8 different Cas variants using T7EI assay, Sanger DNA sequencing, or deep sequencing. Our data indicate that SaCas9 and KKH SaCas9 are highly effective in frogs, which could be used for direct phenotyping in G0 embryos. In contrast, VQR Cas9, xCas9 3.7, SpG Cas9, and SpRY Cas9 were ineffective in X. tropicalis embryos and no activity was detected for iSpyMac Cas9. We also found that LbCas12a/crRNA RNP complexes with paired crRNAs efficiently induced small fragment deletions in X. tropicalis embryos.

Conclusion: SaCas9 and KKH SaCas9 are robust gene targeting tools in X. tropicalis embryos. LbCas12a/crRNA RNP complexes are useful for inducing DNA fragment deletions in frog embryos. These tools expand the CRISPR/Cas genome editing scope in X. tropicalis and increase the flexibility for various genome editing applications in frogs.

SaCas9

KKH SaCas9

LbCas12a

fragment deletion

Xenopus tropicalis

In the past decade, Streptococcus pyogenes Cas9 (SpCas9) has been widely used for genome editing in various species. The clustered regularly interspaced short palindromic repeats (CRISPR)/SpCas9 system requires a NGG PAM at the target site, which obviously limits its editing scope in any given genome [1-3]. To circumvent this limitation, several approaches have been taken. First, a number of Cas9 orthologues from various bacterial genera recognizing different PAMs have been identified and harnessed for genome editing, such as Staphylococcus aureus Cas9 (SaCas9) with the NNGRRT PAM [4], Neisseria meningitidis Cas9 (NmCas9) with the NNAGAAW PAM [5], Streptococcus thermophilus Cas9 (St1Cas9 and St3Cas9) with NNAGAAW and NGGNG PAMs, respectively [6], and Campylobacter jejuni Cas9 (CjCas9) with NNNVRYAC and NNNNRYAC PAM [7]. Second, upon directed evolution of the Cas9 PAM-interacting (PI) domain, various Cas9 variants with altered PAM preferences have been generated, such as VQR SpCas9 (NGA), VRQR SpCas9 (NGCG), xCas9 3.7 (NG, NNG, NGG, GAA, GAT, and CAA), SpG Cas9 (NGN), SpRY Cas9 (NRN and NYN), and KKH SaCas9 (NNNRRT) [8-11]. By replacing the PI domain of SpCas9 with that of Streptococcus macacae Cas9 (SmacCas9), the further engineered iSpyMac Cas9 hybrid can target all adenine dinucleotide (NAA) PAM sequences [12]. Third, members of Class 2 and Type V CRISPR/Cas, such as Cas12a (originally called Cpf1), has also been found to have gene editing capability in some species. Unlike Cas9, Cas12a is a single RNA-guided endonuclease lacking trans-activating crRNA (tracrRNA), which recognizes a short T-rich PAM and induces a staggered DNA double stranded break with a 4-5 nt 5’ overhang. LbCas12a and AsCas12a can be adapted for genome editing in mammalian cells, frog and zebrafish [13-15]. Engineering of Type V-B CRISPR/Cas system member, Cas12b from Bacillus hisashii (BhCas12b), called BhCas12b v4 that utilizes VTTV PAM located at the 5’ end of the target DNA sequence, facilitates robust genome editing in human cell lines and in primary human T cells ex vivo [16]. Recently, the mRNA-active ErCas12a has been reported effective in human cells and zebrafish embryos [17, 18].

The true diploid frog X. tropicalis is an excellent genetic model for both developmental genetics studies and human disease modeling [19]. To date, except for one report on the application of LbCas12a in X. tropicalis[14], the Cas9-mediated targeted gene disruption and integration methods as well as single base editing methods that we and others have established in X. tropicalis are exclusively based on SpCas9[20-25]. Thus, it is highly desirable to expand the CRISPR/Cas genome editing scope in this species.

In this study, we analyzed mutagenic activity of 8 CRISPR/Cas variants in X. tropicalis embryos and found that SaCas9 and KKH SaCas9 are robust tools in frogs. In addition, LbCas12a/crRNA ribonucleoprotein (RNP) complexes showed high efficiency for DNA fragment deletion in X. tropicalis embryos. Adding of these tools definitely expands the CRISPR/Cas genome-editing scope in X. tropicalis.

SaCas9 and KKH SaCas9 have robust gene editing activity in X. tropicalis

SaCas9 and KKH SaCas9 have been proven effective in a number of species including zebrafish [4, 26]. To test their mutagenic effects in X. tropicalis embryos, for each of them, we designed 10 gRNAs targeting 9 different genes. Following the strategy illustrated in Fig. 1, we injected each individual Sa gRNA (50pg/egg) together with either SaCas9 mRNA (300pg/egg) or KKH SaCas9 mRNA (300pg/egg) into fertilized eggs before the first cleavage. For each injected group, 18-24 hours after injection, 60 healthy embryos were collected and divided into two replicates (each 30 embryos) for genome extraction, PCR amplicon library construction, and high-throughput DNA sequencing. The deep sequencing data indicated that vast majority of the target sites tested showed profound targeting efficiency (Fig. 2). For SaCas9, half of the targeted sites (5/10) displayed indel formation frequency above 90% and only 2 appeared inefficient (Fig. 2a, c and Additional file 1: Fig. S1). For KKH SaCas9, the indel efficiency of 8 target sites is more than 60%, and that of other 2 is about 40% and 25%, respectively (Fig. 2b, d and Additional file 1: Fig. S2). Most indels detected are small deletions rarely exceeding 10 bp (Additional file 1: Fig. S1 and Fig. S2). Taken together, these data indicate that both SaCas9 and KKH SaCas9 can effectively edit the genome in X. tropicalis embryos.

VQR Cas9, SpG Cas9, SpRY Cas9, and xCas9 3.7 showed poor mutagenic activity in X. tropicalis embryos

To test more CRISPR/Cas variants for indel induction in X. tropicalis embryos, we designed 8, 15, 4, and 4 gRNAs for SpG Cas9, SpRY Cas9, VQR Cas9, and iSpyMac Cas9, respectively, and checked their mutagenic activity with the T7EI assay. Weak T7EI signals were detected in 2 SpG Cas9 targeting sites, 5 SpRY Cas9 sites, and 3 VQR Cas9 sites. None of the 4 iSpyMac sites displayed discernible T7EI signals (Additional file 1: Fig. S3). For the 10 T7EI positive sites, we further carried out deep sequencing analysis, which confirmed 9 T7EI signals, while the SpRY-best1-NAT site appeared to be a T7EI false-positive one with no indels detected by deep sequencing (Fig. 3a-d and Additional file 1: Fig. S4, Fig. S5 and Fig. S6). Gray value analysis revealed that for each site tested, the low value of the T7EI signal matched well with the corresponding low indel frequency detected by deep sequencing (Fig. 3a-d and Additional file 1: Fig. S3, Fig. S4, Fig. S5 and Fig. S6). Large number of T7EI positive samples have routinely been confirmed by Sanger DNA sequencing in our laboratory. Thus, T7EI assay is a reliable method for detecting targeted gene disruption in our hands.

As xCas9 3.7 has a broad range of PAM compatibility including NG, NNG, NGG, GAA, GAT, and CAA, we designed 143 non-NGG PAM gRNAs targeting 6 genes (tyr, ptf1a/p48, ctcf, apc, kcnj2, and bmp4), 1 NGG PAM gRNA targeting bace2 to cover all these PAMs and assessed their targeting efficacy with the T7EI assay. For NGG PAM sites, we also included 3 gRNAs targeting apc-163, apc-1714, and tbx5-237, which had been proven effective for SpCas9 [24]. Weak T7EI signals were detected at 51 non-NGG sites and 2 NGG sites. Gray value analysis revealed that editing efficiency for most of the positive ones was about 5%. Only 3 sites displayed efficiency slightly over 50% (Fig. 3e and Additional file 1: Fig. S7, Fig. S8a). We selected 4 non-NGG sites and 2 NGG sites with different T7EI signal intensities and carried out a Sanger DNA sequencing verification. The indel frequency detected by Sanger sequencing confirmed the T7EI signals (Fig. 3f and Additional file 1: Fig. S7, Fig. S8c). For NGG sites, the editing efficiency of xCas9 3.7 is much lower than that of SpCas9 (Additional file 1: Fig. S8, and [24]). Collectively, these data indicate that VQR Cas9, SpG Cas9, SpRY Cas9, and xCas9 3.7 do have certain gene editing efficacy with low efficiency in X. tropicalis. But they cannot serve as robust gene editing tools in frogs.

LbCas12a/crRNA RNP complexes are effective for DNA fragment deletion in X. tropicalis

Targeted genome DNA fragment deletion is key to establish animal models of human genetic disorders and to study the function of lncRNA, miRNA, cis-regulatory elements, as well as functionally uncharacterized genome regions. To test the activity of the LbCas12a/crRNA RNP complexes in inducing segmental deletion in X. tropicalis, we designed two pairs of crRNAs targeting two fragments on the tyr locus. For both pairs, gray value analysis of the PCR products on the agarose gel showed that the quantity of the fragments with deletions was roughly the same as that of wild-type fragments, which was confirmed by Sanger DNA sequencing, revealing deletion efficiency of about 50% (Fig. 4a, b). We further designed 4 crRNAs targeting 4 genes (ptf1a/p48, sftpb, tbx5 and ctcf) and tested the mutagenic activity of the LbCas12a/crRNA RNP complexes with the T7EI assay. The data obtained indicate that 3 sites displayed editing efficiency above 50%, of which 2 were further confirmed by Sanger DNA sequencing (Fig. 4c, d). Most indels induced by the LbCas12a/crRNA RNP complexes are deletions that are obviously longer than those induced by SaCas9 or KKH SaCas9(Fig. 4d and Additional file 1: Fig. S1, Fig. S2). Together, these data indicate that LbCas12a/crRNA RNP complexes are highly effective for DNA fragment deletion and indeed robust for gene editing in X. tropicalis.

Phenotyping G0 embryos mutagenized by SaCas9 and KKH SaCas9

Given the high efficiency of gene disruption induced by SaCas9 and KKH SaCas9, knockout phenotypes could be expected in their mutagenized G0 embryos. We have shown that albinism and heart defects could be easily identified upon direct disruption of tyr and tbx5, respectively, in X. tropicalis embryos [21, 24]. Indeed, varying levels of albinism, heart defects, and heart failure related edema depending on the corresponding gene disruption efficiency (effective frameshifts) were observed in SaCas9- or KKH SaCas9-mediated tyr and tbx5 disruption, respectively (Fig. 5; Additional file 1: Fig. S9). These data indicate that SaCas9 and KKH SaCas9 can be used as tools for G0 phenotyping in X. tropicalis.

SaCas9, KKH SaCas9, and LbCas12/crRNA RNP complexes all have low off-target effects in X. tropicalis

To further test the specificity of SaCas9, KKH SaCcas9, and LbCas12/crRNA RNP complexes, we identified genome-wide potential off-target sites with less than five mismatches for 2 SaCas9 targeting sites, 2 KKH SaCas9 targeting sites and 3 LbCas12a targeting sites, with the online software CAS-OFFinder ([27]; Additional file 2: Table S1), of which we selected 23 for T7EI assay (Additional file 2:Table S2). No T7EI signals were detected in these samples (Additional file 2: Table S2, Fig. S10), suggesting low off-target effects of SaCas9, KKH SaCas9, and LbCas12a in X. tropicalis embryos, which is consistent with the SpCas9 activity in frog embryos [21].

In this study, we examined mutagenic activity of several natural Cas nucleases as well as engineered Cas9 variants in X. tropicalis embryos and found that SaCas9 and KKH SaCas9 are comparable to SpCas9, which could be used as tools even for phenotype assessment in G0 embryos (Fig. 5 and Fig. 6). As a known effective tool in frogs, LbCas12a/crRNA RNP complexes are highly effective also for DNA fragment deletion in X. tropicalis embryos. We were unable to detect off-target effects for SaCas9, KKH SaCas9, and LbCas12a with T7EI assay, suggesting low off-target effects with these tools in frog embryos. In contrast, all the SpCas9 variants tested showed very limited activity in X. tropicalis.

The low efficacy of VQR Cas9, SpG Cas9, SpRY Cas9, and xCas9 3.7 in X. tropicalis embryos might be due to temperature. X. tropicalis embryos are normally incubated at 23 ^OC, while all those Cas9 variants were evolved and optimized at 37 ^OC, which may result in reduction of their activity at lower temperatures. It remains to be tested if heat-shock treatment could improve the activity of these variants in X. tropicalis embryos. The natural AsCas12a is very ineffective at low temperature and heat-shock treatment can indeed improve the activity of LbCas12a in zebrafish and X. tropicalis embryos [14]. Of note, in this study we show that without heat-shock treatment, the LbCas12a/crRNA RNP complexes worked well in X. tropicalis embryos at 23 ^OC for both fragment deletion and indel induction, demonstrating the robustness of this tool for routine applications in frogs.

We find that SaCas9 and KKH SaCas9 are robust genome editing tools in X. tropicalis embryos. We also show that LbCas12a/crRNA RNP complexes are effective for DNA fragment deletion in frog embryos. These tools expand the CRISPR/Cas genome editing scope in X. tropicalis and increase the flexibility for various genome editing applications in Xenopus.

Production of Cas9 variants mRNAs, gRNAs, and crRNAs

The human codon-usage optimized VQR-Cas9, SaCas9 and KKH-SaCas9 constructs were purchased from Addgene (Cat. No. 65771，61591, 70708) and sub-cloned into pCS2+ vector. The xCas9 3.7, SpG-Cas9, SpRY-Cas9 and iSpyMac Cas9 were synthesized by IGE Biotechnology (Guangzhou, China) and cloned into pCS2+ vector. The detailed sequences of all Cas9 plasmids are shown in Additional file 2: Table S3. All the Cas9 constructs were linearized with Not I and transcribed with the mMessage mMachine SP6 Kit (Thermo Fisher Scientific) to produce capped Cas9 mRNA, which were purified with the RNeasy Mini Kit (Qiagen) according to the RNA clean protocol.

For all the Cas9 sgRNA transcription, the DNA templates were obtained from a pUC57-T7-gRNA scaffold construct by PCR amplification with the overhang forward primer (Sp gRNA: 5’-CTAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTT-3’, Sa gRNA: 5’-CTAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNNgttttagtactctggaaacagaatc-3’) and universal reverse primer (Sp gRNA: 5’-AAAAAAGCACCGACTCGGTGCCAC-3’, Sa gRNA: 5’-AAAAAATCTCGCCAACAAGTTGAC-3’), transcribed with the Transcript Aid T7 High Yield Transcription Kit (Thermo Fisher Scientific), and purified with the miRNeasy Mini Kit (Qiagen).

LbCas12a protein was purchased from New England Biolabs Inc. LbCas12a crRNA templates were prepared by annealing T7 primer (crRNA-S) with an antisense single strand DNA (crRNA-A) containing a reverse T7 fragment, Cas12a crRNA scaffold, and target sequences, transcribed with the Transcript Aid T7 High Yield Transcription Kit (Thermo Fisher Scientific), and purified with the miRNeasy Mini Kit (Qiagen).

All the target sequences, sequence information for all gRNA and crRNA templates, and PCR primers used for detecting the targeted loci are listed in Additional file 2: Table S4, S5, and S6.

X. tropicalis maintenance, husbandry, and microinjection

X. tropicalis frogs were purchased from Nasco (USA) and bred in an in-house facility. Fertilized eggs and embryos were obtained by hormone-induced mating. Cas9 mRNA (300pg) and sgRNA (50 pg) were co-injected into the animal pole of 1-cell stage embryos in a volume of 2 nl. Cas12a protein (0.75ug/ul) and crRNA (200ng/ul) were assembled 10 min in vitro at 37 ^OC, and were co-injected into the animal pole of 1-cell stage embryos in a volume of 2 nl. During subsequent development, abnormal and dead embryos were sorted out in time.

T7EI (T7 Endonuclease I) assay, Sanger DNA sequencing, and targeted deep-sequencing

Twenty-four hours after microinjection, we randomly pooled 30 (except otherwise indicated) healthy embryos from each injection, extracted genomic DNA, amplified the on-target or off-target regions by PCR (for off-target regions primers, see Additional file 2: Table S2). Then, the disruption efficiency was detected by T7EI assay, Sanger DNA sequencing, or deep-sequencing.

For T7EI assay, PCR products were denatured and annealed under the following conditions: 95°C for 5 minutes, 95-85°C at –2°C/s, 85-25°C at –0.1°C/s, hold at 4°C. The annealed samples were digested with T7EI (NEB M0302L), separated and measured on an 1.5% TAE agarose gel, and quantified using ImageJ software (NIH).

For Sanger DNA sequencing, the purified PCR products were cloned into the pEASY-Blunt Zero Cloning Kit (TransGen Biotech) by Blunt-ended DNA. Single colonies were randomly picked for Sanger DNA sequencing analyses to detect any indel mutations. The targeting efficiency was determined by the ratio of mutant to total colonies sequenced.

For deep-sequencing and data analysis, on-target sites were amplified using 2xPhanta Max Master Mix (Vazyme). Wild-type genomic loci were amplified as a control condition. An VAHTS DNA Clean Beads clean-up step (Vazyme) was performed before pooling and 50 ng of DNA from each condition was used for library preparation. Dual-indexed Illumina deep-sequencing libraries were generated using the VAHTS® Universal DNA Library Prep Kit (Vazyme). All deep-sequencing libraries were sequenced on the Illumina HiSeq X10 platform (paired-end 2 × 150-bp reads). The sequencing data were analyzed using CRISPResso2 [28].

Identification of potential off‑target sites in X. tropicalis

All genomic loci containing up to five mismatches compared with the coding sequence for a given gRNA/crRNA followed by the PAMs sequence were identified by mapping the targeted site to X. tropicalis genome (JGI 9.0) using Cas-OFFinder[27].

Ethics approval and consent to participate

All experiments involving frogs were approved by the Institutional Animal Care and Use Committee at the Southern University of Science and Technology. All animal experiments were conducted in compliance with ethical guidelines.

Consent for publication

Not applicable.

Availability of data and materials

All deep sequencing data generated in this study have been deposited under accession numbers CRA006908 in the National Genomics Data Center of China.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Shenzhen Science and Technology Program (JCYJ20210324120205015).

Authors' contributions

Z.S. and Y.C. conceived the study. Z.S. designed the experiments. H.J. analyzed the deep sequencing data. Z.S. G.L., S.S. and X.Z. performed experiments. Y.C. supervised the project. Z.S. and Y.C. wrote the manuscript with inputs from all the authors.

Acknowledgements

We thank J. Wu for technical assistance and P. Zou for frog husbandry.

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Additional file 2: Supplementary tables.

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Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis

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