Interspecies chimeras serve as valuable models for studying mammalian development and advancing strategies for organ generation. However, xenogeneic barriers-such as mismatched developmental timing, cell adhesion differences, and intercellular competition-restrict the contribution of human pluripotent stem cells (hPSCs) to non-human embryos. Here, we report that Cas13 collateral RNA cleavage can be harnessed to selectively attenuate host cell proliferation to boost donor cell chimerism. In mouse epiblast stem cells (mEpiSCs), Cas13 activation degraded target RNAs and bystander transcripts, producing a reversible decrease in cell proliferation while preserving pluripotency. In co-culture, selective attenuation of mEpiSCs enhanced the competitiveness and survival of wild-type mEpiSCs and hPSCs in both intra- and interspecies settings. In vivo, blastocysts engineered with inducible Cas13 (iCas13) showed increased donor cell integration in chimeric embryos and adult tissues. Together, these findings demonstrate a generalizable strategy to modulate donor-host dynamics and mitigate xenogeneic barriers to interspecies organogenesis.

Creating interspecies chimeras with human pluripotent stem cells (hPSCs) offers a promising strategy for modeling human development and generating donor organs; however, poor human cell integration remains a major barrier. Most existing efforts to improve human chimerism focus on genetically modifying donor hPSCs, while altering the host embryo remains largely unexplored. Using an interspecies PSC competition model, we discovered that RNA innate immunity in "winner" mouse cells drives the competitive elimination of hPSCs. Disrupting RNA-sensing pathways reduced the competitiveness and viability of mouse PSCs, and mouse embryos lacking Mavs-a key gene in RNA innate immunity-led to markedly improved human cell survival and chimerism. We also found that contact-dependent horizontal RNA transfer likely underlies this immune activation. Overall, our study uncovers a previously unrecognized role for RNA innate immunity in cell competition and demonstrates that targeting host immune pathways represents a powerful approach to improve human chimerism in animals.

Interspecies chimeras have served as a crucial tool for understanding the mechanisms of tissue regeneration and repair, offering promising potential to address the global organ shortage crisis. Through a technique known as interspecies blastocyst complementation, researchers can cultivate tissues and organs of one species within the body of another species. This approach involves injecting donor pluripotent stem cells into a host blastocyst that lacks critical developmental genes, allowing the donor cells to compensate for the missing organs or tissues in the host and thereby produce organs derived from the donor species. This review consolidates key findings from studies published in the past 2 years, highlighting advancements in techniques that enable the development of functional organs across species, as well as the remaining challenges.

As the evolutionary ancestor of Cas9 nuclease, IscB proteins serve as compact RNA-guided DNA endonucleases and nickases, making them strong candidates for base editing. Nevertheless, the narrow targeting scope limits the application of IscB systems; thus, it is necessary to find more IscBs that recognize different target-adjacent motifs (TAMs). Here, we identified 10 of 19 uncharacterized IscB proteins from uncultured microbes with activity in mammalian cells. Through protein and ωRNA engineering, we further enhanced the activity of IscB ortholog IscB.m16 and expanded its TAM scope from MRNRAA to NNNGNA, resulting in a variant named IscB.m16*. By fusing the deaminase domains with IscB.m16* nickase, we generated IscB.m16*-derived base editors that exhibited robust base-editing efficiency in mammalian cells and effectively restored Duchenne muscular dystrophy proteins in diseased mice through single adeno-associated virus delivery. Thus, this study establishes a set of compact base-editing tools for basic research and therapeutic applications. Xiao et al. characterize 19 previously unidentified IscB proteins, 10 of which show activity in mammalian cells. They engineer one of the proteins into a robust in vivo genome-editing tool with a broad target-adjacent motif scope and further develop it into a base editor.

Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.

CRISPR–Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications. Several engineered Cas13 variants achieve targeted RNA degradation with minimal collateral effects.

Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (<0.001 relative to Gapdh; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions.

Competitive coevolution between microbes and viruses has led to the diversification of CRISPR-Cas defense systems against infectious agents. By analyzing metagenomic terabase datasets, we identified two compact families (775 to 803 amino acids (aa)) of CRISPR-Cas ribonucleases from hypersaline samples, named Cas13X and Cas13Y. We engineered Cas13X.1 (775 aa) for RNA interference experiments in mammalian cell lines. We found Cas13X.1 could tolerate single-nucleotide mismatches in RNA recognition, facilitating prophylactic RNA virus inhibition. Moreover, a minimal RNA base editor, composed of engineered deaminase (385 aa) and truncated Cas13X.1 (445 aa), exhibited robust editing efficiency and high specificity to induce RNA base conversions. Our results suggest that there exist untapped bacterial defense systems in natural microbes that can function efficiently in mammalian cells, and thus potentially are useful for RNA-editing-based research.

Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant-YE1-BE3-FNLS-that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits. Structural and biochemical insights help engineer a cytosine base editor variant that possesses improved on-target activity with minimal DNA and RNA off-target editing.

RNA knockdown in vivo carries significant potential for disease modelings and therapies. Despite the emerging approaches of CRISPR/Cas9-mediated permanent knock out of targeted genes, strategies targeting RNA for disruption are advantageous in the treatment of acquired metabolic disorders when permanent modification of the genome DNA is not appropriate, and RNA virus infection diseases when pathogenic DNA is not available (such as SARS-Cov-2 and MERS infections). Recently, Cas13d, a family of RNA-targeting CRISPR effectors, has been shown to accomplish robust down-regulation of cellular RNAs in mammalian cells in vitro . Among the various Cas13d subtypes, CasRx (RfxCas13d) showed the most potent RNA knockdown efficiency in HEK293T cells. However, the RNA-targeting activity of Cas13d still needs to be verified in vivo . In this study, the CasRx system was demonstrated to efficiently and functionally knock down genes related to metabolism functions, including Pten, Pcsk9 and lncLstr , in mouse hepatocytes. CasRx-mediated simultaneous knockdown of multiple genes was also achieved by sgRNA arrays, providing a useful strategy to modulate complex metabolism networks. Moreover, the AAV (adeno-associated virus)-mediated delivery of CasRx and Pcsk9 sgRNAs into mouse liver successfully decreased serum PCSK9, resulting in significant reduction of serum cholesterol levels. Importantly, CasRx-mediated knockdown of Pcsk9 is reversible and Pcsk9 could be repeatedly down-regulated, providing an effective strategy to reversibly modulate metabolic genes. The present work supplies a successful proof-of-concept trial that suggests efficient and regulatory knockdown of target metabolic genes for a designed metabolism modulation in the liver.

Unique and multiple repeated sequences for targeting by a single specific sgRNA. (XLSX 9524 kb)

Background: The CRISPR/Cas9 system has become an efficient gene editing method for generating cells carrying precise gene mutations, including the rearrangement and deletion of chromosomal segments. However, whether an entire chromosome could be eliminated by this technology is still unknown. Results: Here we demonstrate the use of the CRISPR/Cas9 system to eliminate targeted chromosomes. Using either multiple cleavages induced by a single-guide RNA (sgRNA) that targets multiple chromosome-specific sites or a cocktail of multiple sgRNAs, each targeting one specific site, we found that a sex chromosome could be selectively eliminated in cultured cells, embryos, and tissues in vivo. Furthermore, this approach was able to produce a targeted autosome loss in aneuploid mouse embryonic stem cells with an extra human chromosome and human induced pluripotent stem cells with trisomy 21, as well as cancer cells. Conclusions: CRISPR/Cas9-mediated targeted chromosome elimination offers a new approach to develop animal models with chromosome deletions, and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes.