Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array

Cpf1 processes its own pre-crRNA array into mature guides with no additional RNase or tracrRNA. An artificial array of four spacers separated by direct repeats from the Francisella novicida CRISPR locus is processed by FnCpf1, AsCpf1 and LbCpf1 alike, and small RNA sequencing shows AsCpf1 cuts at the 5′ end of each direct repeat hairpin — the same cleavage pattern produced by FnCpf1 CRISPR systems expressed heterologously in E. coli. Because each mature crRNA is only 39 nt, one Pol III promoter is enough to drive several guides, whereas multiplexing with Cas9 requires large constructs or co-delivery of multiple plasmids.

The pre-crRNA processing and DNA cleavage activities of AsCpf1 are separable functions of the same protein. Alanine substitution of any of the conserved residues H800, K809, K860, F864 or R790 blocks pre-crRNA processing in vitro while leaving DNA cleavage activity intact, matching the behaviour reported for FnCpf1. The mutants differ in stringency: K809A, K860A, F864A and R790A regain processing at high enzyme concentration or with extended incubation, whereas H800A stays processing-dead regardless of dose or time.

AsCpf1 H800A separates RNA from DNA cleavage in mammalian cells as well as in vitro, producing indel frequencies at the DNMT1 and GRIN2b loci in HEK293T cells identical to wild-type AsCpf1. The separation is not complete across all targets — indel frequencies at VEGFA are higher with wild-type AsCpf1 than with H800A.

AsCpf1 recognizes specific nucleotides in the 5′ flank of the direct repeat stem loop when processing a pre-crRNA array; substituting those nucleotides weakens or abolishes RNA cleavage.

Cpf1's RNase activity attacks the vector that carries it, which constrains lentiviral design: lentiviral virions package a (+) strand RNA copy of the sequence between the long terminal repeats, so a crRNA cassette in that RNA presents a direct repeat that Cpf1 can bind and cleave. Reversing the orientation of the direct repeat in the expression cassette produces (+) strand lentiviral RNA that Cpf1 cannot cleave, and HEK293T cells transduced at an MOI below 0.3 with such a vector show robust indel formation at DNMT1, VEGFA and GRIN2b.

A single customized CRISPR array directs AsCpf1 to edit four genes simultaneously in HEK293T cells — DNMT1, EMX1, VEGFA and GRIN2b — at indel levels comparable to the same guides delivered as individual crRNAs, and small RNA sequencing confirms the processing is autonomous and Cpf1-mediated in mammalian cells. Array architecture matters: of three designs differing in direct repeat and guide length, one dropped EMX1 editing below 2%. Guide position within the array does not matter, since arrays carrying the same guides in different orders give similar indel frequencies.

Delivering several guides as one Cpf1 array edits more loci per individual cell than delivering the same guides as pooled single-guide constructs. Scoring clonal HEK293T colonies by deep sequencing at a threshold of ≥95% indels per locus, array delivery yielded 6.4% of colonies edited at all four targets, 12.8% at three and 48.7% at two, against 2.4%, 3.6% and 11.9% respectively for pooled single-guide transfection.

A dual-AAV system that separates AsCpf1 from a U6-driven three-gene crRNA array achieves multiplex genome editing in the adult mammalian brain. Four weeks after stereotactic injection of the two vectors at a 1:1 ratio into the mouse hippocampal dentate gyrus, co-transduction efficiency reaches about 75%, and FACS-purified transduced nuclei carry roughly 23%, 38% and 51% indels at Mecp2, Nlgn3 and Drd1, with about 17% of transduced neurons edited at all three loci at once. GFP fused to the KASH nuclear transmembrane domain, encoded on the array vector, is what makes the nuclei of targeted cells sortable.

Multiplex AAV-delivered AsCpf1 editing disrupts both alleles in the majority of edited cells in vivo: about 47% of all sorted dentate gyrus nuclei, corresponding to roughly 87% of Drd1-edited cells, carry biallelic modification of the autosomal gene Drd1.