Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

Although multiplex gene editing is possible with Cas9, it requires relatively large constructs or simultaneous delivery of multiple plasmids , both of which are problematic for multiplex screens or in vivo applications. In contrast, Cpf1 only requires one Pol III promoter to drive several small crRNAs (39nt per crRNA). We confirmed that Cpf1 alone is sufficient for array processing[ using an artificial CRISPR pre-crRNA array consisting of four spacers separated by direct repeats (DRs) from the CRISPR locus of Francisella novicida (FnCpf1) and two Cpf1 orthologs with activity in mammalian cells, Acidaminococcus Cpf1 (AsCpf1) and Lachnospiraceae Cpf1 (LbCpf1) (). Small RNAseq showed that AsCpf1 cleavage products correlate to fragments resulting from cuts at the 5’ end of DR hairpins, identical to the cleavage pattern we observed in E.coli heterologously expressing FnCpf1 CRISPR systems[ ().

We further validated these results by generating AsCpf1 mutants that are unable to process arrays. Guided by the crystal structure of AsCpf1[, we mutated five conserved amino acid residues likely to disrupt array processing (H800A, K809A, K860A, F864A, and R790A) [. All mutations interfered with pre-crRNA processing but not DNA cleavage activity in vitro (), an effect that was also observed for FnCpf1[. AsCpf1 recognizes specific nucleotides at the 5’ flank of the DR stem loop. Substitution of these nucleotides weakens or abolishes RNA cleavage (). Dosage tests with the five AsCpf1 mutants revealed that mutants K809A, K860A, F864A, and R790A show pre-crRNA processing when used at high concentration () or for extended incubation times (), but H800A was inactive regardless of dose and time.

We next tested if this mutant retains DNase activity in human embryonic kidney (HEK) 293T cells using three guides. Insertion/deletion (indel) frequency at the DNMT1 and GRIN2b loci were identical between wild-type and H800A AsCpf1, whereas indel frequencies at the VEGFA locus were higher in cells transfected with wild-type AsCpf1, demonstrating that the RNA and DNA cleavage activity can be separated in mammalian cells ().

Cpf1 mediated RNA cleavage needs to be considered when designing lenti-virus vectors for simultaneous expression of nuclease and guide (). Lenti virions carry a (+) strand RNA copy of the sequence flanked by long terminal repeats (LTR), allowing Cpf1 to bind and cleave at DR sequences. Hence, reversing the orientation of the DR is expected to result in (+) strand lenti RNAs not susceptible to Cpf1 mediated cleavage. We designed a lenti vector encoding AsCpf1 and a crRNA expression cassette. We transduced HEK293T cells with a MOI (multiplicity of infection) of <0.3 and analyzed indel frequencies in puromycin selected cells 10 days post infection. Using guides encoded on a reversed expression cassette targeting DNMT1, VEGFA, or GRIN2b resulted in robust indel formation for each targeted gene ().

We leveraged the simplicity of Cpf1 crRNA maturation to achieve multiplex genome editing in HEK293T cells using customized CRISPR arrays. We chose four guides targeting different genes (DNMT1, EMX1, VEGFA, and GRIN2b) and constructed three arrays with variant DR and guide lengths for expression of pre-crRNAs (). Indel events were detected at each targeted locus in cells transfected with array-1 or -2. However, the crRNA targeting EMX1 resulted in indel frequencies of <2% when expressed from array-3. Overall, array-1 performed best, with all guides showing indel levels comparable to those mediated by single crRNAs (). Furthermore, small RNAseq confirmed that autonomous, Cpf1-mediated pre-crRNA processing occurs in mammalian cells (). Using arrays with guides in different orders resulted in similar indel frequencies, suggesting that positioning within an array is not crucial for activity ().

To confirm that multiplex editing occurs within single cells, we generated AsCpf1-P2A-GFP constructs to enable fluorescence-activated cell sorting (FACS) of transduced single cells () and clonal expansion. We used next generation deep sequencing (NGS) to compare edited loci within clonal colonies derived from cells transfected with either pooled single guides or array-1. Focusing on targeted genes edited at every locus (indels ≥95%) shows that multiplex editing occurs more frequently in colonies transfected with array-1 (6.4% all targets, 12.8% three targets, 48.7% two targets) than in pooled transfection (2.4% all targets, 3.6% three targets, 11.9% two targets).

We next tested multiplex genome editing in neurons using AsCpf1. We designed a gene-delivery system based on adeno-associated viral vectors (AAVs) for expression of AsCpf1. We generated a dual vector system in which AsCpf1 and the CRISPR-Cpf1 array were cloned separately (). We constructed a U6 promoter-driven Cpf1 array targeting the neuronal genes Mecp2, Nlgn3, and Drd1. This plasmid also included an green fluorescent protein (GFP) fused to the KASH nuclear transmembrane domain [ to enable FACS of targeted cell nuclei [.

We first transduced mouse primary cortical neurons in vitro and observed robust expression of AsCpf1 and GFP-KASH one week after viral delivery. SURVEYOR nuclease assay on purified neuronal DNA confirmed indel formations in all three targeted genes (). Next, we tested whether AsCpf1 can be expressed in the brains of living mice for multiplex genome editing in vivo. We stereotactically injected our dual vector system in a 1:1 ratio into the hippocampal dentate gyrus (DG) of adult male mice. Four weeks after viral delivery we observed robust expression of AsCpf1 and GFP-KASH in the DG (). Consistent with previous studies [, we observed ~75% co-transduction efficiency of the dual viral vectors (). We isolated targeted DG cell nuclei by FACS () and quantified indel formation using NGS. We found indels in all three targeted loci with ~23%, ~38%, and ~51% indel formation in Mecp2, Nlgn3, and Drd1, respectively (). We quantified the effectiveness of biallelic disruption of the autosomal gene Drd1 and found ~47% of all sorted nuclei (i.e. ~87% of all Drd1-edited cells) harbored biallelic modifications (). Next, we quantified the multiplex targeting efficiency in single neuronal nuclei. Our results show that ~17% of all transduced neurons were modified in all three targeted loci (). Taken together, our results demonstrate the effectiveness of AAV-mediated delivery of AsCpf1 into the mammalian brain and simultaneous multi-gene targeting in vivo using a single array transcript.

Taken together, these data highlight the utility of Cpf1 array processing in designing simplified systems for in vivo multiplex gene editing. Although multiplex gene editing is possible with Cas9, it requires relatively large constructs or simultaneous delivery of multiple plasmids [, both of which are problematic for multiplex screens or in vivo applications. In contrast, Cpf1 only requires one Pol III promoter to drive several small crRNAs (39nt per crRNA). Hence, this system has the potential to simplify guide RNA delivery for many genome editing applications where targeting of multiple genes is desirable.