New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing.
ABSTRACT: CRISPR-Cas has proven to be the most versatile genetic tinkering system of our time, predominantly as a precision genome editing tool. Here, we demonstrate two additions to the repertoire of CRISPR's application for constructing donor DNA templates: CRISPR-CLONInG and CRISPR-CLIP. CRISPR-CLONInG (CRISPR-Cutting and Ligation Of Nucleic acid In vitro via Gibson) was devised to enable efficient cut-and-paste of multiple complex DNA fragments by using CRISPR-Cas9 as a digestion alternative with precision and exclusivity features, followed by joining the digested products via Gibson Assembly, to construct double-stranded DNA and adeno-associated virus (AAV) donor vectors rapidly without cloning scars. CRISPR-CLIP (CRISPR-Clipped Long ssDNA via Incising Plasmid) was devised as a DNA clipping tool to retrieve long single-stranded DNA (lssDNA) efficiently from plasmid, up to 3.5?kbase, which can be supplied as the donor template for creating genetically engineered mice via Easi-CRISPR. We utilized two different Cas types (Cpf1 and Cas9n) to induce two distinct incisions at the respective ends of the lssDNA cassette junctions on the plasmid, yielding three independent single-stranded DNA units of unique sizes eligible for strand separation, followed by target strand clip-out through gel extraction. The retrieval of the lssDNA donor circumvents involvements of restriction enzymes and DNA polymerase-based steps. Hence, it not only retains sequence fidelity but also carries virtually no restriction on sequence composition, further mitigating limitations on the current Easi-CRISPR method. With the add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo protospacer adjacent motif, CRISPR-CLIP can be facile and applicable to generate lssDNA templates for any genomic target of choice. Additionally, we demonstrate robust gene editing efficiencies in the neuroblastoma cell line, as well as in mice attained with the AAV and lssDNA donors constructed herein.
Project description:Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (<i>Danio rerio</i>). Our study demonstrated that an ?200 base-pair sequence encoding a composite tag can be efficiently "knocked-in" into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the <i>sox3</i>, <i>sox11a</i>, and <i>pax6a</i> genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3' homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the <i>sox3</i> and <i>pax6a</i> knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited <i>sox3</i>, <i>sox11a</i>, and <i>pax6a</i> knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5' and 3' junctions indicated that knock-in allele frequencies were higher at the 3' side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos.
Project description:Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%. Large gene deletions and insertions are also obtained using an all-in-one plasmid consisting of FnCpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3N+4 or 3N+2 days. A proof-of-concept, codon saturation mutagenesis at G149 of γ-glutamyl kinase relieves L-proline inhibition using Cpf1-assisted ssDNA recombineering. Thus, CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas9 system.
Project description:The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
Project description:Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method.We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors.lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.
Project description:Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (?3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1-crRNA complex is an effective DNA-free genome-editing tool for plant genome editing.
Project description:CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
Project description:While CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing.We systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform precise gene targeting efficiently across multiple genomic loci using single-stranded oligodeoxynucleotide (ssODN) donor templates with homology arms as short as 17 nucleotides. Strikingly, the two Cpf1 nucleases exhibit a preference for ssODNs of the non-target strand sequence, while the popular Cas9 enzyme from Streptococcus pyogenes (SpCas9) exhibits a preference for ssODNs of the target strand sequence instead. Additionally, we find that the HDR efficiencies of Cpf1 and SpCas9 can be further improved by using asymmetric donors with longer arms 5' of the desired DNA changes.Our work delineates design parameters for each CRISPR-Cas system and will serve as a useful reference for future genome engineering studies.
Project description:The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.
Project description:The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases.