CRISPR/Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We have analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observe that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning and Cas9 off-target binding are not major determinants. We additionally identified truncated and 5′ mismatch-containing sgRNAs as efficient alternatives to canonical sgRNAs. Based on these results, we created a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures the sequence features affecting Cas9/sgRNA activity in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos-3′-UTR fusion can generate maternal-zygotic mutants, increase viability and reduce somatic mutations. Together, these results provide novel insights into the determinants that influence Cas9 activity and a framework to identify highly efficient sgRNAs for genome targeting in vivo.

Cellular transitions require dramatic changes in gene expression that are supported by regulated mRNA decay and new transcription. The maternal-to-zygotic transition is a conserved developmental progression during which thousands of maternal mRNAs are cleared by post-transcriptional mechanisms. Although some maternal mRNAs are targeted for degradation by microRNAs, this pathway does not fully explain mRNA clearance. We investigated how codon identity and translation affect mRNA stability during development and homeostasis. We show that the codon triplet contains translation-dependent regulatory information that influences transcript decay. Codon composition shapes maternal mRNA clearance during the maternal-to-zygotic transition in zebrafish, Xenopus, mouse, and Drosophila, and gene expression during homeostasis across human tissues. Some synonymous codons show consistent stabilizing or destabilizing effects, suggesting that amino acid composition influences mRNA stability. Codon composition affects both polyadenylation status and translation efficiency. Thus, the ribosome interprets two codes within the mRNA: the genetic code which specifies the amino acid sequence and a conserved "codon optimality code" that shapes mRNA stability and translation efficiency across vertebrates.

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.

Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to induce RNA degradation in yeast, plants and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-Cas13d is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that both zygotically-expressed and maternally-provided transcripts are efficiently targeted, resulting in an 80% average decrease in transcript level and the recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish and mouse embryos. Altogether our results demonstrate that CRISPR-Cas13d is an efficient knock-down platform to interrogate gene function in animal embryos..

The awakening of the genome after fertilization is a cornerstone of animal development. However, the mechanisms that activate the silent genome after fertilization are poorly understood. Here, we show that transcriptional competency is regulated by Brd4-and P300-dependent histone acetylation in zebrafish. Live imaging of transcription revealed that genome activation, beginning at the miR-430 locus, is gradual and stochastic. We show that genome activation does not require slowdown of the cell cycle and is regulated through the translation of maternally inherited mRNAs. Among these, the enhancer regulators P300 and Brd4 can prematurely activate transcription and restore transcriptional competency when maternal mRNA translation is blocked, whereas inhibition of histone acetylation blocks genome activation. We conclude that P300 and Brd4 are sufficient to trigger genome-wide transcriptional competency by regulating histone acetylation on the first zygotic genes in zebrafish. This mechanism is critical for initiating zygotic development and developmental reprogramming.

SUMMARY Proper functioning of an organism requires cells and tissues to behave in uniform, well-organized ways. How this optimum of phenotypes is achieved during the development of vertebrates is unclear. Here, we carried out a multifaceted and single-cell resolution screen of zebrafish embryonic blood vessels upon mutagenesis of single and multi-gene miRNA families. We found that embryos lacking particular miRNA-dependent signaling pathways develop a vascular trait similar to wild type, but with a profound increase in phenotypic heterogeneity. Aberrant trait variance in miRNA mutant embryos uniquely sensitizes their vascular system to environmental perturbations. We discovered a previously unrecognized role for specific vertebrate miRNAs to protect tissue development against phenotypic variability. This discovery marks an important advance in our comprehension of how miRNAs function in the development of higher organisms.

This protocol describes how to generate and genotype mutants using an optimized CRISPR-Cas9 genome-editing system in zebrafish (CRISPRscan). Because single guide RNAs (sgRNAs) have variable efficiency when targeting specific loci, our protocol starts by explaining how to use the web tool CRISPRscan to design highly efficient sgRNAs. The CRISPRscan algorithm is based on the results of an integrated analysis of more than 1000 sgRNAs in zebrafish, which uncovered highly predictive factors that influence Cas9 activity. Next, we describe how to easily generate sgRNAs in vitro, which can then be injected in vivo to target specific loci. The use of highly efficient sgRNAs can lead to biallelic mutations in the injected embryos, causing lethality. We explain how targeting Cas9 to the germline increases viability by reducing somatic mutations. Finally, we combine two methods to identify F heterozygous fish carrying the desired mutations: (i) Mut-Seq, a method based on high-throughput sequencing to detect F founder fish; and (ii) a polymerase chain reaction-based fragment analysis method that identifies F heterozygous fish characterized by Mut-Seq. In summary, this protocol includes the steps to generate and characterize mutant zebrafish lines using the CRISPR-Cas9 genome engineering system.

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